Abstract Background The aim of this study is to assess the clinical effectiveness of the 3-step hand hygiene technique (3-HT) compared to the 6-step hand hygiene technique (6-HT,World Health Organization 6-step technique) using alcohol-based hand rub (ABHR). Methods A randomized controlled crossover trial was conducted from November to December 2023 in 10 wards of a tertiary A-level hospital according to CONSORT guidelines. The 240 healthcare workers (HCWs) were randomly divided into the 3-HT intervention group or the 6-HT control group. The trial was conducted in two stages, and the effectiveness of each indicator in the two groups was compared after a washout period of two weeks. Results Compared to the 6-HT, the 3-HT has demonstrated significant superiority in all indicators of hand hygiene compliance (HHC) as well as the accuracy rate of hand hygiene (HH) practices. The total HH time of the 3-HT and 6-HT were [median 16.00, interquartile range (IQR) (15.00-20.00) second (s)] vs [median 32.50, IQR (30.00-40.00)s], respectively (P < 0.05). The reduction factor (RF) of bacterial colony counts did not differ and the colony-forming units were not significantly different. Bacillus, Staphylococcus and Micrococcus were detected before and after the use of handrubs. HCWs preferred the 3-HT over the 6-HT. There were no significant difference in healthcare-associated infections (HAIs) rate between the two techniques. Conclusions The 3-HT was significantly superior to the 6-HT in terms of the HH eligibility rate, compliance, HH time. The safety and feasibility of the 3-HT were verified by assessing microorganism count.