The invention discloses a structure detection method of a protein cryoelectron microscopy density map, which belongs to the field of bioinformatics. The method comprises the following steps of: 1) selecting an atom matching conditions in an atom model to indicate the atom model; 2) selecting a key point in a protein cryoelectron microscopy density map to be inspected and adding the key point to a set M; 3) for a point g in the set M, calculating a point g which is closest to the point p in a set M and is used as a corresponding point to the point p, and adding the point g in a set G; 4) calculating a rotation quaternion q and a translation vector t of the atom model according to the sets P and G; 5) calculating a rotation matrix R according to the rotation quaternion q, and calculating the position of each rotated atom coordinate according to R and t, so as to obtain the matching result of the atom model and the density map, and 6) obtaining the structure of the protein cryoelectron microscopy density map to be detected according to the matching result meeting the requirements. The method has the advantages that the robustness is good, the data model is not required to be coincident with density data, the calculated amount is small, and programming is easy to realize.