This study proposes a modular floating biofilm reactor (MFBR) for in situ nitrogen removal from slightly polluted water in rivers using enriched indigenous microorganisms. Its main structure is a 60 cm × 60 cm × 90 cm rectangular reactor filled with hackettens. After a 96-day startup, the removal efficiencies of ammonia-N and total N (TN) reached 80% and 25%, respectively, with a hydraulic retention time (HRT) of 10 h, whereas those in a control reactor (without biofilm) were only 4.9% and 0.2%, respectively. The influences of HRT and dissolved oxygen (DO) were also investigated. As a key factor, HRT significantly affected the removal efficiencies of ammonia-N and TN. When HRT was close to the actual value for a river studied (2.4 min), the removal efficiencies of ammonia-N and TN were only 8.7% and 3.1%, respectively. Aeration increased the concentration of DO in water, which enhanced nitrification but inhibited denitrification. When HRT was 2.4 min, aeration intensity was 20 L/min; the ammonia-N and TN removal rates were 9.5 g/(m2·d) and 11.3 g/(m2·d), respectively. The results of microbial community analysis indicated that the microorganisms forming the biofilm were indigenous bacteria. The findings demonstrated a concept-proof of MFBR, which may be evaluated in scaling up investigation for developing a new methodology for nitrogen removal from slightly polluted surface water in plain river networks.