Objective To explore the application effects of cluster nursing management in the collection of blood samples from neonatal two diseases (phenylketonuria, congenital hypothyroidism) screening. Methods A total of 2336 neonates who were screened for two diseases in our hospital from January 2017 to December 2017 were selected as subjects. 1145 neonates born between January 2017 and June 2017 were set as control group, and 1191 neonates born between July 2017 and December 2017 were set as study group. Study group was given cluster nursing management, and control group was given routine nursing. The blood collection time, pain degree, qualified rate of blood sampling, success rate of one-time blood sampling, incidence rate of blood stasis and blood cake quality were compared between the two groups. Results The blood collection time in study group was shorter than that in control group, and the pain score was lower than that in control group, and the qualified rate of blood collection, success rate of one-time blood collection and the proportion of blood cake>1 cm were higher than those in control group, and the incidence rate of blood stasis and the proportion of 0.7-1 cm blood cake were lower than that those in control group (P<0.05). Conclusion Cluster nursing management in the collection of blood samples from neonatal two diseases screening can improve the success rate, qualified rate and quality of blood collection, and can reduce the pain and shorten the blood collection time.