Daily hassles are frequent yet a danger to our health. When daily events are challenging and appraised as an obstruction, we perceive them as stressors, and they typically trigger negative emotions, particularly when we fail to cope with them. Mindfulness can enhance individuals’ emotion regulation when facing stressors and can help individual to have more toleration and acceptance to negative sensations and feelings. Moreover, researchers found mindfulness predicted lower stress perceptions and fewer negative emotions. It is possible that the dynamic relation between perceived stress and negative emotions in daily life may vary among individuals with different levels of dispositional mindfulness. Individuals with external locus of control usually have a poor performance in stress coping process. However, externals tend to take less actions when facing stressors, which means that they have more chances to switch into the “being mode” (vs. “doing mode”) of mindful state, compared to those internals. Therefore, it is possible that mindfulness may be more helpful for the externals. The current study investigated the relation between perceived stress and negative emotions in daily life measured by ambulatory assessment, and the moderations of mindfulness and locus of control were also tested. A total of 95 college students completed the Mindful Attention Awareness Scale (MAAS) and the Rotter’s Locus of Control Scale (LOCS). Then they participated in an ambulatory assessment procedure in which they reported their perceived stress and negative emotions (including anxiety, depression, anger, fatigue, discomfort and overall negative emotion) in daily life twice a day for 14 consecutive days. Multilevel model was conducted to analyze the data by HLM 7.0. Results indicated that (1) at within-person level, perceived stress at time t positively predicted all negative emotions at time t+1; (2) mindfulness was associated with lower prediction of perceived stress on negative emotions (including anxiety, fatigue, discomfort and overall negative emotion) in daily life; (3) internal locus of control was associated with lower prediction of perceived stress on negative emotions (including anxiety, fatigue, discomfort and overall negative emotion) in daily life; (4) the moderation of mindfulness in the prediction of perceived stress on negative emotions (including anxiety, fatigue, discomfort and overall negative emotion) for externals were stronger than that for internals. In summary, the current study revealed the dynamic association between perceived stress and negative emotions in daily life, and explored how this association varies in individuals with different mindfulness levels, which indicated the protection of mindfulness in stress process. Furthermore, the current study found that mindfulness may be more helpful for externals (compared with internals) to improve stress coping. This finding suggests that mindfulness-based interventions may be more useful for individuals with internal locus of control in stress reduction. Future study should generalize the findings to clinical samples and should investigate whether mindfulness-based interventions can change the dynamic relation between perceived stress and negative emotions in daily life and whether the externals can get more benefits from the interventions.