Uneven illumination, vignetting, and stray light lead to the undesirable decrease of response uniformity of high-throughput fluorescence imaging systems, which brings difficulties to subsequent image processing and data analysis. Here, we propose a flat-field correction method using array targets specially designed for fluorescence microscopy. Based on several dedicated correction sources and the response model, the proposed algorithm performs step-by-step correction to eliminate nonuniformities caused by stray light, high-frequency inhomogeneity, and low-frequency response inhomogeneity. For verification, the correction method is applied to a high-throughput microscopic imaging system with several time-delay-integration detectors. Experimental results show that the proposed flat-field correction method is effective and practical.