A new analytical approach utilizing a biofilm reactor (BFR) for rapid online determination of biochemical oxygen demand (BOD) was proposed and experimentally validated. The BFR was fabricated via a cultivation process using naturally occurring microbial seeds from locally collected wastewaters. The resultant BFR displays a remarkable rate of biodegradation towards a wide spectrum of organic substrates, capable of degrading over 20% of biodegradable organic substrates within 100 s. More importantly, the BFR exhibits a superior indiscriminative biodegradation feature, enabling a precise prediction of BOD values of total biodegradable organics based on experimentally determined BOD values from partial degradation processes without a need for on-going calibration. The proposed approach was systematically validated using a range of individual organic substrates, their mixtures, synthetic samples and wastewaters. Highly significant linear correlations between the BFR and the standard BOD5 methods were obtained from diversified synthetic samples (r = 0.988, p = 0.000, n = 45) and wastewaters (r = 0.983, p = 0.000, n = 40). Near unity slope values of the principal axis of the correlation ellipse were obtained from all tested samples, suggesting both methods were essentially measuring the same BOD value. The reported method could be a useful online monitoring tool for determination of biodegradable organic pollutants.