Objectives The optimized scheme of applying long-acting progesterone in the program technology of synchronized estrus-timed insemination in large-scale farms was studied to increase the pregnancy rate of mating postpartum dairy cows. Method The mixed farming of Holstein dairy cows and Holstein-Friesian hybrid cattle was selected to set up 6 experimental groups. The 45th day postpartum Holstein cows (Group A) and Holstein cows (Group B) were used to optimize the program by adding long-acting progesterone on day 0 and pregnant horse serum on day 7 based on the program technology of 0-7-9 synchronized estrus-timed insemination. The pregnancy rate of different breeds under the same insemination procedure was compared. The pregnancy rate of different breeds of Holstein cows (Group C) and Holstein cows (Group D) under natural estrus conditions was compared. The experimental Holstein cows (Group E) were farmed with an optimized program of applying long-acting progesterone in synchronized estrus-timed insemination, while the control Holstein cows (Group F) were farmed with a basic program. The pregnancy rate of the same breed under different treatments was compared. Results The optimized program of applying long-acting progesterone in synchronized estrus-timed insemination in experimental groups A and B resulted in a 2.17 percent increase in pregnancy rate in Holstein-Friesian hybrid cattle compared to that in Holstein dairy cows. The pregnancy rate of postpartum Holstein-Friesian hybrid cattle in groups C and D under natural estrus increased by 17.31 percent compared to that in Holstein dairy cows. The pregnancy rate of the Holstein-Friesian hybrid cattle in the E and F groups with optimized program of applying long-acting progesterone in synchronized estrus-timed insemination was 17.35 percent higher than that with the basic program. Conclusions The application of long-acting progesterone on the basis of program technology of applying long-acting progesterone in synchronized estrus-timed insemination can effectively improve the pregnancy rate of postpartum dairy cows.