An evaluation method of autofocus function based on spatial domain is presented. Four features are proposed to evaluate the function on computation time and sharpness of the focus curve. An optimum can be filtrated from many classic functions. Entropy function is the evaluating result through experiments on the developed autofocus system of micro assembly workcell. A novel focus searching algorithm——positioning algorithm (PA) which is different from hill climbing searching alorithm (HCSA) and Fibonacci Searching Algorithm (FSA) is put forward. Setting up the relation between defocus value and focus function value is key factor of the algorithm. Three methods: polynomial fitting, Gaussian function fitting and cubic spline interpolation fitting are presented to realize PA. Then an autofocus system comprising entropy function and PA is set up. Experimental results indicate that focusing precision can reach 6.992 μm and one focusing processing can be done within 3.4 s. It proves that the evaluation method and the PA can realize autofocus accurately and quickly.