Iterative Shrinkage Thresholding Algorithm (ISTA) often uses fixed iteration step to solve the dynamic optimization problem of depth from defocus, which leads to poor convergence efficiency and low accuracy of reconstructed microscopic 3D shape. A method based on gradient estimation of acceleration operator and secant linear search, called Fast Linear Iterative Shrinkage Thresholding Algorithm (FL-ISTA), was proposed to optimize ISTA. Firstly, the acceleration operator, which consists of the linear combination of the current and previous points, was introduced to reestimate the gradient and update the iteration point during each iteration process. Secondly, in order to change the restriction of the fixed iteration step, secant linear search was used to determine the optimal iteration step dynamically. Finally, the improved algorithm was applied to solve the dynamic optimization problem of depth from defocus, which accelerated the convergence of the algorithm and improved the accuracy of reconstructed microscopic 3D shape. Experimental results of reconstructed standard 500 nm grid show that compared with ISTA, FISTA (Fast ISTA) and MFISTA (Monotohy FISTA), the efficiency of FL-ISTA was improved and the depth from defocus decreased by 10 percentage points, which is closer to the scale of standard 500 nm grid. Compared with ISTA, the Mean Square Error (MSE) and average error of microscopic 3D shape reconstructed by FL-ISTA were decreased by 18 percentage points and 40 percentage points respectively. The experimental results indicate that FL-ISTA can effectively improve the convergence rate of solving the dynamic optimization problem of depth from defocus and elevate the accuracy of the reconstructed microscopic 3D shape.