Confocal laser scanning microscopy(CLSM) is an invaluable tool for a wide range of investigations in the biological and medical sciences. A type construction of laser con-focal scanning and fluorescent detection system was provided. A polygon mirror and a galvanometer scanner were used to implement x-y scan. The relay optical system was designed based on commercial lens with optimized consideration to achieve small scan spot size, large Field-of-View and high efficiency. Fluorescence was detected by a photomultiplier tube with excellent signal-to-noise ratio. The scan system control and image acquisition were designed with an optimized scan velocity and sample clock. All these measures could reduce the blurring effect in the whole imaging process and improve the resolution. The analysis results show that the design of the CLSM is reasonable and all components achieve an optimized consideration for in-vivo scanning imaging; this type of CLSM are easily accessible and can be upgraded according to optical requirements; the performance is comparable to available commercial products, but is superior in many aspects of cost, flexibility and versatility.