Dichroic mirrors, as core optical elements, are widely used in biomedical testing instruments based on the principles of absorbance detection, fluorescence detection and Raman detection. In this paper, the application principle of dichroic mirrors for biomedical detection was introduced, and some key technical indexes for high-performance dichroic mirrors were proposed according to their application characteristics. With the equivalent layer substitution method, a dichroic mirror film system of high-performance based on two conventional materials was designed. Using the plasma assisted reactive magnetron sputtering technology, the dichroic mirror film system with 146 layers was coated, and a kind of dichroic mirror with some important technical indicators such as high steepness (<1%), depolarization (polarization separation <3.5nm), wide range of high transmission (T>96% range > 300nm) was manufactured. The influence of wavefront distortion of dichroic mirror on imaging was described in detail. Our dichroic mirrors can meet the performance of low transmitted wavefront distortion (TWD<λ/4) and low reflected wavefront distortion (RWD<λ/2). The manufactured dichroic mirror products showed satisfying application results when used in high-end medical testing instruments with fine imaging requirements.