Abstract: Disclosed is a composition for increasing pregnancy rate in ruminants exposed to ovsynch and timed artificial insemination, and a preparation method and application thereof. The effective component of the composition is melatonin with a concentration of 10-20 mg/mL. The present disclosure proves through animal experiments that the use of the composition of the present disclosure in combination with the last injection of GnRH in an ovsynch and timed artificial insemination protocol for a ruminant can regulate the secretion of reproductive hormones in an animal body, increase the levels of luteinizing hormone (LH) and progesterone in pregnant females, and improve ovum quality and facilitate ovulation, thus increasing the pregnancy rate in ruminants exposed to ovsynch and timed artificial insemination.