This study proposes a digital holographic autofocusing method employing the gray-level co-occurrence matrix (GLCM) and Lanczos interpolation. Key limitations in conventional autofocus methods, such as low clarity of focusing curves, low sensitivity near the focal point, and long computation time, are addressed. The proposed method leverages the GLCM of the reconstructed amplitude and Lanczos interpolation to compute contrast for digital holography autofocusing. Numerical and experimental simulations of resolution plate and experimental focusing results on gastric wall section cells confirm that the proposed method significantly outperforms conventional approaches. Specifically, compared with existing four typical methods, the proposed method maintains equivalent focusing accuracy while achieving an average 78-fold increase in focus curve clarity and an average 41-fold increase in sensitivity. Furthermore, compared with the existing two typical methods, it achieves an approximately 27.8% reduction in computation time.