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We evaluated the contribution of net biological effects (photosynthesis + respiration + decomposition) to the carbonate parameters and air–water CO2 fluxes in Tokyo Bay, Ise Bay, and Osaka Bay in Japan. The carbonate parameters (fugacity of CO2, total alkalinity, and dissolved inorganic carbon) were measured mainly by cargo ships traveling between Japan and other countries. We used the measurement data from three inner bays and surrounding outer bays in Japan along with reference data from previous studies for complementary analysis. We found that (a) the inner and outer bays in this study were strong annual atmospheric CO2 sinks, (b) the annual biological effect on the air–water CO2 fluxes was about 6%–27% of the measured CO2 fluxes, and it affected the seasonal variation of the CO2 flux, and (c) the biological effect was largest in Tokyo Bay, and almost the same in Ise and Osaka Bays. The intensity of the biological effect corresponded mainly with nutrient concentrations, which seemed to be controlled by the wastewater treatment in urbanized areas around the bays. Our results suggest that labile carbon/nutrient ratio of wastewater should be a major consideration for evaluating the biological effect on the carbon cycle in urbanized bays, which will continue to expand globally. Plain Language Summary: We analyzed the biological effect (photosynthesis, respiration, and decomposition) on air–water CO2 exchange in Tokyo Bay, Ise Bay, and Osaka Bay in Japan using data from cargo‐ship measurements and previously published reports. We concluded that (a) bay water strongly absorbs atmospheric CO2, (b) biological effects accounted for 6%–27% of the evaluated CO2 absorption and had significant effects on its seasonal variation, and (c) the biological effects seemed to be mediated mainly by the low degradable carbon/nutrient ratio in wastewater. This study should improve our understanding of the carbon flow in urbanized coastal areas, which are expanding globally. Key Points: Urbanized bays in Japan are reportedly annual atmospheric CO2 sinks but comprehensive measurements are fewWe quantified biological effects on carbonate parameters and CO2 flux using an empirical relationship with riverine and oceanic endmembersThe biological effect was regulated mainly by nutrient concentrations related to wastewater treatment [ABSTRACT FROM AUTHOR]