Non-point nitrate pollution in groundwater has been accelerated by agricultural development, but sustainable nitrogen removal is a challenge because of its wide distribution and negative side effects. Surface agricultural practices (SAPs), which are demonstrably effective in driving the downward infiltration of dissolved organic carbon (DOC), have not been well explored for their potential to enhance nitrate attenuation in groundwater. Therefore, a combination of soil column and groundwater incubation experiments was performed to investigate the carbon and nitrogen responses to different SAPs (manure fertilization, lucerne planting, and straw return). The soil column experiment showed that SAPs promoted DOC and reduced nitrate leaching into groundwater, and straw treatment witnessed the highest DOC leaching flux (252.71 g m−2 yr−1) and lowest nitrate leaching flux (9.51 g m−2 yr−1). The groundwater incubation experiment showed that leachates from the straw treatment displayed the best denitrification-enhancement performance, with the highest NO3−-N reduction efficiency (92.93%) and rate (1.627 mg/day), N2 selectivity (99.78%), and net nitrogen removal (0.09 mg). Furthermore, Fourier transform ion cyclotron resonance mass spectrometry confirmed that CHOS molecules with lower double bond equivalents (0–5) and larger carbon numbers (10–15) were more accessible to denitrifiers. This study provides a new path for the sustainable control of non-point source nitrate pollution.