Chinese is a tone language; the semantic information of Chinese is attached with segmental information(vowel、consonant) and also supra-segmental information(tone). Although we have known the effect of the phonological information, it is not clear of the specific effect course between the two. We used the eye tracking method, and conducted two experiments to examine the effect of Chinese bisyllable supra-segmental information on semantic activation. Experiment 1 investigated the effect of Chinese bisyllable tone information on semantic activation. the task of the participants is to choose one word which is related in meaning with the word they heard, there were four Chinese bisyllable words in the screen, the four words were in the four corners, one of them is related in meaning with the heard word, one of them is changed both the tone of initial character and the last character, the rest of the two words are the word frequency controlled words of related word and tone changed word, the heard word and the screen are presented at the same time. The eye track shows that the tone changed word has more fixation and gaze time than its controlled words, it means the tone really effects on semantic activation, but the effects of the tone of initial character or last character, which is bigger? We designed experiment 2 to investigate this problem. In experiment 2, the tone changed word had two levers, one was only changed the tone of the initial character, and the other was only changed the tone of the last character, we found that the effects of the tone of initial character is bigger than the last character. This result shows the initial tone has more important effect than the last tone on semantic activation. In the two experiments, we didn’t find the word frequency effect. Word frequency effect is that the high word frequency word can make the react time shorter, and the low word frequency word can make the reaction time longer. We think the factor of word frequency in this research is very complicated, because it related with the task, and the personal use habit, and we also didn’t control the inner word frequency between the two characters. Many factors make the result didn’t find the word frequency effect. Based on the above studies’ results analysis and discussions, the main conclusions were drawn as follows: (1) on semantic activation of Chinese bisyllable, when changed the tone of initial character or last character, it constraints of semantic activation, and the initial character changed has lager effect than last. This result supports the Cohort theory. (2)The fixation count and fixation time is more than control word, but there is no word frequency effect. (3)The process of Chinese tone is similar to Chinese vowel and consonant, effect the semantic activation isolate. Many researches of phonological coding of words were done, but there exists controversies over the problem of cerebral hemisphere processing lateralization for the language function. The future research is to investigate the time course of Chinese segmental information and supra-segmental information by using eye movements recording and ERP technique, analyze its source of the cerebral neural activity.