To examine the interference effects of a 532 nm nanosecond pulse laser on two representative image sensors, namely charge-couple device (CCD) and complementary metal-oxide semiconductor (CMOS), experimental studies were conducted in accordance with the testing methodology outlined in ISO-21254, under both atmospheric and vacuum conditions. The impact of varying pulse numbers within a 50 ms exposure duration was systematically compared and analyzed alongside the interference effects observed in different environments. The findings indicate that both CCD and CMOS are significantly influenced by the 532 nm nanosecond laser, resulting in optical saturation phenomena. Additionally, CCD demonstrates “reverse saturation” and “saturation crosstalk”. As the number of applied pulses increases, the quantity of saturated pixels exhibits a linear growth relative to laser energy density, with an accelerated growth rate corresponding to an increase in pulse count. Notably, CCD displays superior performance against interference effects in vacuum compared to atmospheric conditions; conversely, CMOS shows more pronounced interference effects when tested under atmospheric conditions but possesses greater resilience against 532 nm nanosecond pulse laser disturbances than its CCD counterpart. These research outcomes provide valuable insights for selecting detectors suitable for practical application environments.