Laser scanning confocal microscopy (LSCM) is a high-resolution imaging device. It uses the principle of “conjugate imaging”, the image quality obtained is much higher than that of ordinary fluorescence microscopy. LSCM has two pairs of pinholes, namely illumination pinhole and detection pinhole, which have different function respectively and are the key to realize conjugate imaging. Although the size and position of the illumination pinhole are generally fixed, LSCM can obtain high-quality images by adjusting the size of the detection pinhole. However, there is a lack of knowledge about the link between parameter of pinhole and the higher quality picture for LSCM. Therefore, this paper reveals the principle of pinhole in LSCM and its relationship with resolution. Furthermore, based on immunofluorescence image analysis for specific proteins expression in white matter of mouse spinal cord, we found that optimization for pinhole play an important role in LSCM image quality improving. This study would provide important reference for LSCM imaging analysis.