Biomass of three species of macrophytes, Pistia stratoides (water lettuce), Eichhornia crassipes (water hyacinth), and Phragmites australis (common reed) as well as their combination, were added into baffled subsurface-flow constructed wetlands (BSFCWs) as carbon source for the treatment of nitrate-laden wastewater. Nitrogen removal performance and responses of substrate enzyme activities (nitrate reductase, dehydrogenase, CM-cellulase, β-glucosidase, urease, and protease) were investigated and assessed in the present study. Nitrogen removal was significantly improved by all of the biomass (averaging 0.27 g TN·m−2 day−1 for control and 2.15–2.80 g TN·m−2 day−1 for the experimental systems), with best performance achieved by P. stratiotes. Dissolved oxygen and oxidation-reduction potential values in the biomass-added systems were significantly lower, beneficial for nitrate reduction. Effluent organic carbon content was low due to the low amount of biomass added, but quick decline of nitrate removal efficiency and nitrite accumulation were observed. All of the enzyme activities were notably enhanced by the biomass addition, especially in the initial phase after addition, which might be significant for decomposition and utilization of the macrophyte biomass for denitrification and the enhancement of nitrogen removal of the BSFCWs.