The integration of near-infrared genetically encoded reporters (NIR-GERs) with photoacoustic (PA) imaging enables visualizing deep-seated functions of specific cell populations at high resolution, though the imaging depth is primarily constrained by reporters’ PA response intensity. Directed evolution can optimize NIR-GERs’ performance for PA imaging, yet precise quantifying of PA responses in mutant proteins expressed in E. coli colonies across iterative rounds poses challenges to the imaging speed and quantification capabilities of the screening platforms. Here, we present self-calibrated photoacoustic screening (SCAPAS), an imaging-based platform that can detect samples in parallel within 5 s (equivalent to 50 ms per colony), achieving a considerable quantification accuracy of approximately 2.8% and a quantification precision of about 6.47%. SCAPAS incorporates co-expressed reference proteins in sample preparation and employs a ring transducer array with switchable illumination for rapid, wide-field dual-wavelength PA imaging, enabling precisely calculating the PA response using the self-calibration method. Numerical simulations validated the image optimization strategy, quantification process, and noise robustness. Tests with co-expression samples confirmed SCAPAS’s superior screening speed and quantification capabilities. We believe that SCAPAS will facilitate the development of novel NIR-GERs suitable for PA imaging and has the potential to significantly impact the advancement of PA probes and molecular imaging.