Objective To explore the influencing factors and improving measures of hand hygiene among healthcare workers. Methods From June to August 2016, several healthcare workers from clinical departments and nosocomial infection control department in Chengdu were selected by purposive sampling method. Data was collected by individual in-depth interviews, and was three-rank coded by Nvivo 8 software based on Grounded Theory. Results After three-rank coding, 6 important influencing factors were generalized which were re-categorized into 3 levels: personal cognition, behavior capacity and social support. At the last, the whole framework of the theory was constructed through core coding. Conclusion In clinical practice, we should take reasonable measures to strengthen the training of hand hygiene, improve the hand hygiene facilities, strengthen supervision and management, and effectively improve the implementation rate of hand hygiene.