Objective To study the effect of different sampling methods on microbial monitoring results of dialysis water at the connection between dialysis machine and water supply circuit. Methods Dialysis water at the connection between 54 dialysis machine and water supply circuit in a hospital from January 2018 to December 2020 was sampled, according to different sampling methods, samples were divided into three groups: single-person sampling group (January-December 2018), double-person sampling group (January-December 2019) and sampling-valve sampling group (January-December 2020). Bacteria, endotoxin and sampling time of dialysis water in three groups were monitored. Results The endotoxin contents of dialysis water in single-person sampling group, double-person sampling group, and sampling-valve sampling group were (0.24±0.33), (0.11±0.09), and (0.06±0.03) EU/mL respectively, the total colony numbers of bacterial culture were (19.43±23.13), (9.17±14.73), and (1.26±1.62) CFU/mL respectively, and the sampling time were (196.50±16.52), (144.17±11.50), and (55.02±5.87) seconds respectively, the overall difference was significant (all P < 0.05). Multiple comparisons among groups showed that differences were all significant (all P < 0.05). In sampling-valve sampling group, sampling time was the shortest (55.02 seconds), average content of endotoxin was the lowest (0.06 EU/mL), and the colony number of bacterial culture was the lowest (1.26 CFU/mL). The qualified rates of endotoxin content in dialysis water of single-person sampling group, double-person sampling group and sampling-valve sampling group were 66.67%, 92.59% and 98.15% respectively, and qualified rates of bacterial culture were 77.78%, 94.44% and 100% respectively, difference of the overall comparison was significant, except that there was no significant diffe-rence between double-person sampling group and sampling-valve sampling group, pairwise comparison of the other groups were all significantly different (all P < 0.016 7). Conclusion Sampling of dialysis water at the connection between dialysis machine and water supply circuit through sampling-valve can effectively ensure qualified rate and accuracy of sampling results, improve sampling efficiency, ensure dialysis quality and safety, and effectively avoid the increase of department cost.