A three-dimensional microscopic measurement based on gradient variance is proposed for focus evaluation to achieve high-precision focusing. Building upon the traditional Brenner evaluation function, this method incorporated information on changes in the grayscale gradient along the vertical and diagonal directions. Additionally, by capturing richer image edge details, it enhances the sensitivity of the focus evaluation function, thereby improving the accuracy of three-dimensional microscopic measurements. Experiments with a step sample having a nominal height of 1 mm were conducted, yielding a relative measurement error of 0.49% (relative to the standard value) for the proposed method, outperforming traditional focus evaluation methods with an error of 1.22%. Furthermore, for the evaluation function, the proposed method exhibits a clarity ratio and a sensitivity with values of 2.4412 and 318.45, respectively.