The invention discloses an activating agent for indigenous microorganisms of heavy oil reservoir and application thereof, and belongs to the field of oil exploitation. The activating agent comprises, by mass, 1.0-1.5% of rhamnolipid solution, 0.2-0.3% of fucto oligosaccharide, 0.3-0.45% of mlasses, 0.10-0.15% of ammonium chloride, 0.08-0.15% of yeast cream, 0.10-0.15% of ptassium posphate mnobasic, 0.15-0.25% of sodium acetate, 0.05-0.10% of polyacrylamide and the balance oil reservoir production water. The percentage mass content of rhamnolipid is equal or greater than 3.5% in the rhamnolipid solution; the relative molecular weight of the polyacrylamide is from 20 million to 25 million; each milliliter of oil reservoir production water contains at least 104 indigenous microorganisms. The activating agent can effectively improve the recovery efficiency of the heavy oil reservoir, and it is predicted that the final recovery efficiency can be increased by 6.4%.