The invention provides an indigenous microorganism activating agent screening method under high temperature and high pressure system and application thereof. The method includes the steps that activating agents to be screened are mixed with crude oil, after system culture is carried out at the temperature of 50-65 DEG C and pressure of 11.5-13.5 MPa, the total bacterium concentration is measured, and the concentration of hydrocarbon oxidizing bacteria, emulsifying functional bacteria, sulfate reducting bacteria and methanogens is measured; the activating agents to be screened are mixed with the crude oil, and crude oil characteristics and characteristics of the interface between methanogens fermentation liquor and the crude oil are evaluated after system culture is carried out at the temperature of 50-65 DEG C and pressure of 11.5-13.5 MPa; the physical simulation oil displacement effect of the activating agents to be screened is evaluated; all the indexes are calculated and scored, and scores obtained from all the indexes are added to obtain the comprehensive scores of the agents to be screened. The screening method aims at a high temperature and high pressure system and has the characteristics of being comprehensive, systematic and high in operability and specificity, the blank of effect evaluation of existing activating agents under the high temperature and high pressure system is filled up, and an effective basis is provided for improving the field application effect of the indigenous microorganism oil recovery technique.