Wastewater-derived dissolved organic nitrogen (DON) should be minimized by municipal wastewater treatment plants (MWWTPs) to reduce its potential impact on receiving waters. Solids retention time (SRT) is a key control parameter for the activated sludge (AS) process; however, knowledge of its impact on effluent DON is limited. This study investigated the effect of SRT on the bioavailability, fluorescent components, and molecular characteristics of effluent DON in the AS process. Four lab-scale AS reactors were operated in parallel at different SRTs (5, 13, 26, and 40 days) for treatment of primary treated wastewater collected from an MWWTP. Results showed the positive effect of prolonged SRT on DON removal. AS reactors during longer SRTs, however, cannot sequester the bioavailable DON (ABDON) and occasionally contribute to greater amounts of ABDON in the effluents. Consequently, effluent DON bioavailability increased with SRT ( R2 = 0.619, p < 0.05, ANOVA). Analysis of effluent DON fluorescent components and molecular characteristics indicated that the high effluent DON bioavailability observed at long SRTs is contributed by the production of microbially derived nitrogenous organics. The results presented herein indicate that operating an AS process with a longer SRT cannot control the DON forms that readily stimulate algal growth.