Objective To understand the current status and influencing factors of humanistic care knowledge, attitude, and practice among nursing assistants, and to provide a reference for developing training measures for humanistic care. Methods A convenient sampling method was used to select 181 nursing assistants from three hospitals as the subjects of the survey from August to November 2024. A general information questionnaire and the Knowledge, Attitude and Practice (KAP) Questionnaire on Humanistic Care for Nursing Assistants were used for the investigation. Results The total score and the standard scores for knowledge, attitude, and behavior dimensions of the KAP Questionnaire on Humanistic Care for Nursing Assistants were (85.91±4.77), (82.76±8.64), (84.97±6.43), and (87.76±5.30), respectively. Multiple linear regression analysis showed that the level of the hospital where they work, educational level, and family location are influencing factors of the level of humanistic care knowledge, attitude, and practice among nursing assistants (all P<0.05). Conclusion The overall level of humanistic care knowledge, attitude, and practice among nursing assistants is good, but there is a relative lack of knowledge in humanistic care. Healthcare managers, training companies, and relevant departments should provide corresponding training to improve their level of humanistic care knowledge, attitude, and practice.