Objective To investigates the awareness of medical staff on prevention of mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV) . Methods Between April and July 2014, health workers from towns and cities of Liangshan Prefecture participated in the class of prevention of HIV PMTCT for key areas in Sichuan province. Those participants, together with some other health workers from the same area whose daily work was related to prevention of PMTCT, were selected as the study subjects. PMTCT prevention knowledge survey questionnaire was given out for the investigation, and chi-square test was used for statistical analysis. Results We retrieved altogether 447 (out of 450) valid questionnaires. Of all the respondents, 94.18% were aware of proper time for HIV antibody testing and routes of transmission in the perinatal period, 95.30% knew the strategy of applying as early as possible antiviral drugs during pregnancy, and 86.58% learned to know when to give the first dose to the newborns and avoidance of breast-feeding. However, the awareness of relevant applied knowledge was relatively low. The total awareness rate of medical workers from women and children’s hospitals were significantly better than that of medical workers from general hospitals (P<0.05), and differences of the awareness depended on different specialties, professional titles and working age (P<0.05). Conclusions The majority of medical personnel in the surveyed area have grasped basic knowledge of PMTCT of HIV, but the ability of applying relevant knowledge precisely, promptly and standardly in daily practices needs to be improved. We recommend that, in addition to strengthening routine training on knowledge of PMTCT of HIV, the mastering and practice of key technical intervention strategies should be followed up and evaluated, especially for those from general hospitals. And it is also important to give training and after-training assessment based on different specialties.