In this paper, the electrochemiluminescence (ECL) of sulfur quantum dots (SQDs) in a potassium persulfate cathodic co-reactant was studied. Based on the selective quenching of the ECL emission from the SQDs by β-nicotinamide adenine dinucleotide (NADH), an ultrasensitive ECL biosensor with NADH as an important parameter was established for the highly sensitive and selective detection of total bacterial count (TBC). A linear response between the ECL intensity and the NADH concentration in the range of 1 pM to 10 μM was obtained, thus achieving a detection limit of 1 pM. As the content of NADH in cells is positively correlated with the TBC, a sensor has been successfully applied to detect the TBC in actual water samples with a good recovery rate of 103-107 CFU mL-1. This study provides a green and feasible method for TBC detection in the environment.