The concern about wastewater effluent toxicity has motivated the innovation of enhancement technologies on sulfur-based denitrification biofilter in recent years. Electrolysis is a common technology to reduce or remove toxic pollutants. However, the effect of electrolysis on simultaneous total nitrogen (TN) removal and toxicity reduction in sulfur-based denitrification biofilter has not been reported yet. Herein, for the first time, this study investigated the synergistic effects of electrolysis-induced TN removal and toxicity reduction of secondary effluent of dyeing wastewater containing 20 μg/L of nonylphenol (NP), at different carbon to nitrogen ratios (C/N) in several sulfur-based denitrification biofilters. All of the biofilters achieved the denitrification rate of 300.15 g∙N/m3∙d during the stabilization period at C/ N = 5. The CSAHD (ceramisite and sulfur as filters) biofilter had highest TN removal rate to achieve the denitrification rate of 257.46 g∙N/m3·d at C/ N = 2. Siderite and dolomite both facilitated TN removal efficiency by 9.3%–12.6% under low C/N ratio and acted as the buffer agent in biofilters. Toxicity characteristic leaching procedure (TCLP) test showed that the amount of leached heavy metals was lower than the concentration limit standard of USEPA. Electrolysis did not promote the removal of TN, however, it could reduce NP concentration and increase the biotoxicity relative inhibition rate of effluent by 12.5%–167%, and affect the functional microbial community structure. Our work clarified some misunderstandings about the application of electrolysis-based strengthening technology and enlightened the future development of simultaneous TN removal and toxicity reduction of dyeing wastewater. Unlabelled Image • Simultaneous TN removal and toxicity reduction in electrolysis-AHD biofilters for the first time. • Siderite and dolomite both increase TN removal efficiency by 9.3%–12.6% under low C/N ratio. • Electrolysis does not promote the removal of TN. • Electrolysis increases biotoxicity relative inhibition rate by 12.5%–167%. • Electrolysis could alter functional microbial community structure. [ABSTRACT FROM AUTHOR]