Cumulative evidence suggests that practice has significantly dissociable influences on stimulus and response conflicts in the congruency tasks (such as the Stroop and Flanker tasks). However, the effects of practice on stimulus and response conflicts in a typical Flanker task are unclear. To address this question, in the present study, behavioral and electroencephalography (EEG) data were recorded from twenty-nine healthy human participants while they were performing a 2:1 (two targets are mapped into one response key) letter Flanker task in a practice design. The stimuli consisted of capital letters (‘N’, ‘H’, ‘K’, and ‘F’). In each trial, five horizontal-arranged capital letters were presented in Times New Roman font with a central target letter flanked by two distractor letters on each side. The participants were instructed to respond to the central target letter (1) ‘N’ or ‘H’ by pressing the “2” key with the left forefinger and (2) ‘K’ or ‘F’ by pressing the “9” key with the right forefinger. Participants performed a block of sixteen trials prior to the completion of seven experimental blocks. In the experimental blocks, the first and last served as the pre- and post-practice blocks consisting of 192 trials each; the practice stage consisted of 1200 trials separated into 5 blocks of 240 trials each. To reveal the practice-related behavioral and spectral characteristics, behavioral and EEG analyses only focused on the response time (RT), error rates, and oscillatory magnitudes in pre- and post-practice blocks. Modulations of EEG activities in the time-frequency domain were represented as an event-related spectral perturbation (ERSP) that was estimated using a continuous wavelet transform (CWT). To distinguish between phase-locked and non-phase-locked EEG responses, the phase-locking value (PLV) was respectively calculated in pre- and post-practice stages, for each trial type of each participant. Based on the previous investigations, we will test the hypotheses that (1) the effects of practice on RT in the letter Flanker task may be different from those in the Stroop and color-word Flanker tasks, and (2) practice may affect the frontal theta and alpha activities. The results show that RT significantly reduced with practice; however, stimulus and response conflicts did not vary after practice. It is likely that practice enhanced the links between stimuli and responses for each trial type in the same way and, therefore, shortened RT to the same degree for each trial type, and did not affect stimulus and response conflicts. Brain oscillatory results are consistent with the RT patterns and showed that (1) the frontal theta (4–7 Hz, 0.52–0.82 s) activity significantly declined for each trial type after practice, which suggests the enhanced links between stimuli and responses after practice, and (2) the frontal alpha (8–11 Hz, 0.52–0.82 s) activity was constant for both stimulus and response conflicts with practice, suggesting a stable mechanism of stimulus and response conflict processes in the letter Flanker task. Therefore, we speculate that the pattern of theta activity may reflect the task characteristics, and that the modulations of the alpha magnitudes may reflect an innate mechanism of stimulus- and response-conflicts, which is task-specific and, therefore, does not be affected by the vast practice. Altogether, these findings demonstrate stable behavioral and neural oscillatory characteristics of stimulus and response conflicts in a typical Flanker task.