In this paper,based on the depth from defocus method,we design an automatic focusing optical path,incl uding laser,collimation lens,focusing lens and CCD camera.ZEMAX software is utilized to simulate the optical path of the focusing process,through which the error is estimated within 1mm,when the di stances of focus target are 5.62cm,7.38cm,14.02cm,16.88cm and 20.94cm.In this basis,the focal optical path is constructed and simulated.Meanwhile the diameters of the light spots are measu red through a camera and analyzed by MATLAB software,which shows an error within 1cm.Both simulati on and experimental results demonstrate the feasibility of the depth from defocus approach to fulfill the a uto-focusing process,in consideration of the error of the guide reading,the limited camera pixel and th e unstable laser power. The subsequent work will replace the manual slide rail with the displacement pla tform,and the program will control the moving position of the lens and realize automatic focusing.The research work provides a technical reference for improving the portable and high sensitive narcotics fluo rescence detection system.