The invention discloses a rotating total internal reflection microscopy device with a feedback function. The device comprises a laser device, a two-dimensional scanning galvanometer, a scanning lens, a collimating lens, a dichroic mirror, a beam splitter, a dichroic mirror, a microscopic field lens, a total reflection microobjective and a sample which are arranged in sequence along an optical path, a light intensity position detector located on the reflection optical path of the beam splitter, a computer and a CCD used for acquiring the sample and emitting fluorescent light, wherein the light intensity position detector is used for collecting illuminating light which is emitted by the laser device and reflected by the beam splitter to obtain a first light spot and collecting sample light formed after total reflection of the position of the sample to obtain a second light spot, and the computer is used for obtaining total reflection illumination angle and evanescent wave penetration depth through feedback according to the position information of the first light spot and the second light spot. The invention further discloses a rotating total internal reflection microscopy method with the feedback function. Through synchronous feedback control of the sample and the microobjective, it can be guaranteed that the sample is located on the optimal illumination face and the optimal imaging face, and illumination uniformity and imaging resolution are higher.