This paper presents an improved autofocus method for human red blood cell images in a microscope. The products of the sum modulus difference and the real-valued fast Fourier transform function are multiplied to obtain an improved sharpness evaluation using the properties of a Gaussian function. It is superior to traditional evaluations in terms of unimodality, steepness, and sensitivity. A new quantitative criterion is proposed to represent the ability of sharpness evaluation against noise. An adaptive focus window with great robustness is proposed that can reduce the computation cost and adverse effects of the background. The better performances of the proposed algorithms are all proved by experiment results, and they can help to find the quasi-focus position more quickly and accurately.