In this manuscript, a sandwich electrogenerated chemiluminescence (ECL) biosensor for galactosyltransferase (Gal T) activity analysis based on a graphitic carbon nitride (g-C3N4) nanosheet interface and polystyrene microsphere-enhanced responses is described. The carboxylated g-C3N4 nanosheet was firstly coated on the GC electrode surface, and then the bovine serum albumin conjugated with N-acetylglucosamine (GlcNAc-BSA) was further modified on the g-C3N4 modified GC electrode. In the presence of Gal T and UDP-Gal as a coreactor, galactose was conjugated to GlcNAc-BSA, and the ECL signal of the GlcNAc-BSA conjugated g-C3N4 nanosheet modified electrode decreased. After the adsorption of Artocarpus integrifolia lectin (AIA) conjugated polystyrene microsphere (PSM) nanoprobes through the specific interaction between galactose and AIA, the ECL signal decreased significantly due to the poor conductivity of the nanoprobes, which inhibited the electron transfer on the electrode interface, leading to high sensitivity toward Gal T activity analysis. The detection limit of 7 x 10(-5) U mL(-1) was obtained. The proposed ECL sensor has been applied to Gal T activity analysis in serum samples as well as Gal T activity expression in different cell lines, and may be a novel tool to for glycosyltransferase activity evaluation and inhibition in clinic diagnostics. This strategy can also be extended to detect and monitor other glycosyltransferases.