Objective To investigate the training needs and expectations of primary health workers in Pukou District, and provide optimized countermeasures for their on-the-job training programs. Methods In August 2020, a questionnaire survey was used to investigate 428 primary health workers in Pukou District, Nanjing. The survey content included their basic situation, training needs and expectations. Results Among the 428 primary health workers surveyed, 68.9%(295/428) expressed their need for training. The primary motivation of 59.7% (231/387) of primary health workers to participate in the training is to “strengthen basic skills and improve the medical level”; 97.6% (40/41) of those who have not participated in the training have never participated in the training because “the training location is not suitable”; 50.2% (215/428) were interested in receiving training in “new technology and new knowledge”; 43.5% (186/428) expected training in the form of “specialist training”. Conclusions The training needs of primary health workers in Pukou District are not high, and they expect to receive practical training. By mobilizing the training enthusiasm of the low-demand workers, and formulating the training plan for primary health workers based on their needs and expectations, the training efficiency of primary health workers can be effectively improved.