To meet the high precision requirements of structured light 3D measurement, a subpixel extraction method for laser stripes based on cubic spline interpolation and Gaussian fitting is proposed. Preprocessing including region of interest extraction and Gaussian filtering was first performed on the laser stripe images. The laser stripes were then thinned to obtain the initial center points using morphological methods. The normal direction of the laser stripes was obtained using the five-point fitting method. Fitting points were taken at subpixels in the normal direction of the laser stripes. The pixel values of subpixel coordinates were obtained by cubic spline interpolation. Finally, Gaussian fitting was performed to obtain the subpixel centers of the laser stripes. Experiments show that compared with the previous algorithm, this method has higher extraction accuracy and stability.