Due to the similar chemical structure of carbonyl sulfide (COS) and carbon dioxide (CO₂) as well as the co-absorption of COS and CO₂ by plants, it is possible to employ COS as a proxy for estimating the gross primary productivity (GPP). However, the response of COS and CO₂ fluxes to environmental factors varies between different plants. Two typical subtropical tree species, namely Pinus massoniana and Cunninghamia lanceolata were selected, two nitrogen levels, and three soil water gradients were set in this study. Gas samples were collected in sample bags with dynamic headspace cuvettes methods, and the COS concentration inside was determined by preconcentrator-GC-MS. The photosynthetic parameters of plants was measured simultaneously. The results showed that P. massoniana and C. lanceolata could assimilate COS, with the rates of 39.58-127.27 pmol m^-2 s^-1 and 0.81-66.92 pmol m^-2 s^-1, respectively. Generally, COS absorption by plants could be promoted by nitrogen application. Except for the significant effect of nitrogen application on the COS flux of P. massoniana (P < 0.05), soil moisture, nitrogen application and their interactions had no significant effect on COS and CO₂ fluxes (F_COS and F_CO2) and their ratios of F_COS and F_CO2 for P. massoniana and C. lanceolata. With nitrogen application, the COS absorption by P. massoniana could be promoted at high soil moisture, while at low soil moisture for C. lanceolata. Under moderate soil water and high soil water conditions, the COS fluxes of P. massoniana and C. lanceolata were positively correlated with stomatal conductance. The linear fitting results showed that the COS fluxes of plants were extremely significantly positively correlated with the CO₂ fluxes (P < 0.01), with the F_COS/F_CO2 value of 1.48×10^-6 and 1.01×10^-6 for P. massoniana and C. lanceolata, respectively. Moderate soil moisture could increase F_COS/F_CO2 value for P. massoniana, while nitrogen application increased F_COS/F_CO2 value under low soil water conditions, and the value decreased under high soil water conditions for C. lanceolata. Low and high soil moisture increased the vapor pressure deficit of P. massoniana, which promoted the reduction of stomatal conductance and reduced the net photosynthetic rate. Nitrogen application increased the stomatal conductance of C. lanceolata under low and high soil moisture conditions and thus enhanced net photosynthetic rate. The results not only have important implications for further understanding of the effects of regional nitrogen deposition and precipitation on tree COS flux and F_COS/F_CO2, but also provide regional data for modelling and estimating gross primary productivity.