In this study, the influence of the working distance (WD) on strain measurement under a laser scanning microscope and a way to achieve precise focus were investigated by the scanning moiré method. Experimental results showed that the strain measurement has a good repeatability at a fixed WD. Scanning moiré fringes were clearly observable when the WD variation range was within 0.9% of the given WD of the used objective lens. The relationship of the measured strain error and the WD difference was approximately linear, and the greatest strain error was near 700 με. Fortunately, 2D moiré fringes were distinct only in a very narrow range, i.e., the WD difference was less than 0.1% of the given WD, and the greatest strain error was less than 100 με. 1D moiré fringes in the y direction, 2D moiré fringes in the both x and y directions, and 1D moiré fringes in the x direction became distinct alternately along with the WD change. Consequently, we suggest to use 2D moiré fringes for microscale strain measurement in each focusing process to reduce the errors caused by the WD variation. Moreover, a single-shot 2D moiré image is useful to measure the strain distributions in both two directions quickly and simply, and there is no need to rotate the sample or scanning lines and scan twice as in the conventional way.