The invention relates to the technical field of biology, in particular to Shh pathway regulation and control biological rhythm and related application thereof. Specifically, the invention provides application of a Hedgehog pathway inhibitor and an SMO inhibitor in regulating the biological rhythm and treating diseases related to the biological rhythm. Preferably, the Hedgehog pathway inhibitor comprises the following components: PF-5274857, Mebendazole, HPI-4, SANT-1, Taladegib, Grasdegib, Cyclopamine, Itracazole, GANT61, JK184, Robotnikinin, Vismodegib, Purmorphamine, and Sonidegib Phoshate, and the Hedgehog pathway inhibitor comprises the following components: PF-5274857, the Mebendazole, the HPI-4, the SANT-1, the Taladegib, the Grasdegib, the Cyclopamine, the Itracazole, the GANT61, the JK184, the Robotnikinin, the Vismodegib, the