Soil respiration (Rs) is one of the major components controlling the carbon budget of terrestrial ecosystems. Aerated irrigation has been proven to increase Rs compared with the control, but the mechanisms of CO2 release remain poorly understood. The objective of this study was (1) to test the effects of irrigation, aeration, and their interaction on Rs, soil physical and biotic properties (soil water-filled pore space, temperature, bacteria, fungi, actinomycetes, microbial biomass carbon, cellulose activity, dehydrogenase activity, root morphology, and dry biomass of tomato), and (2) to assess how soil physical and biotic variables control Rs. Therefore, three irrigation levels were included (60%, 80%, and 100% of full irrigation). Each irrigation level contained aeration and control. A total of six treatments were included. The results showed that aeration significantly increased total root length, dry biomass of leaf, stem, and fruit compared with the control (p < 0.05). The positive effect of irrigation on dry biomass of leaf, fruit, and root was significant (p < 0.05). With respect to the control, greater Rs under aeration (averaging 6.2% increase) was mainly driven by soil water-filled pore space, soil bacteria, and soil fungi. The results of this study are helpful for understanding the mechanisms of soil CO2 release under aerated subsurface drip irrigation.