The invention discloses a medium-high temperature oil preserved bacterium population detection gene chip and application thereof. A gene group of a sample to be detected serving as a template is amplified by a labeled primer group; the obtained amplified product is hybridized with a probe on a biological chip; and the components of an oil preserved bacterium population are determined according to a hybridization result. The sample is an oil developing fluid material for forecasting and containing a target nucleotide sequence; oil preservation comprises natural oil preservation and artificial simulation oil preservation; the primer group comprises one group or more than one group of common primers or specific primers, can amplify a nucleotide segment including an oil preserved microorganism identification base and has a length of 5 to 30nt; and the biological chip comprises a plurality of types of probes, wherein the probe can be specifically combined with a detected microorganism molecule. The invention provides a quick, accurate and high-flux tool to the study on the component, the structure, the functional activities and the like of the oil preserved environment bacterium population and provides theoretical basis and technical support for the field implementation effect.