This paper aims to investigate the effects of ultrasonic duration, power and frequency on the removal of marine microfouling on polymethyl methacrylate (PMMA) surface on the basis of Box-Behnken design and response surface analysis, by analyzing surface morphology characterization and fouling area ratio, and through the fitting equation and experimental verification, thus providing some theoretical and experimental data supports for the application of ultrasonic antifouling technology in microfouling control of underwater window in marine optical instruments. During this period, the microfouling on PMMA surface was firstly constructed by dynamic cultivation in natural seawater. Secondly, the effect laws of ultrasonic duration, power and frequency on the removal of microfouling on PMMA surface were investigated by means of the laser confocal microscope (CLSM), and by single factor experiment and three-factor three-level Box-Behnken design. Further, the ultrasonic antifouling interaction among ultrasonic frequency, ultrasonic time and ultrasonic power was obtained by response surface analysis, and the regression equation of ultrasonic parameters on antifouling effect was established. Finally, the optimum ultrasonic parameters applied for removing fouling were given based on the actual working conditions. The results show that with the increase of ultrasonic frequency, the antifouling effect decreases, but increases with the increase of ultrasonic power and ultrasonic duration. Compared with ultrasonic duration and ultrasonic power, ultrasonic frequency has a more significant effect on the microfouling removal of PMMA surface. The regression equation can well predict the antifouling effect under different ultrasonic parameters, so it can be used for the selection and design of ultrasonic working conditions according to practical requirements of optical instruments. When the ultrasound duration is 5 min, ultrasound power is 40 W and ultrasound frequency is 40 kHz, the antifouling effect on the PMMA surface can reach to 98.63% after the seawater dynamic cultivation for 96 h. Therefore, it is concluded that, the regression equation of ultrasonic parameters on ultrasonic antifouling effect is established base on response surface analysis, which is helpful to the selection of the suitable ultrasound conditions, thus making the ultrasonic antifouling technology applied in the removal of microfouling on the surface of underwater windows of marine optical instruments.