As one of the glycosyltransferase involved in protein glycosylation, beta-1,4-galactosyltransferase (Gal T) plays an important role in the cellular process and progression of cancer. Here, using the bovine serum albumin conjugated N-acetylglucosamine (GlcNAc-BSA) as a receptor to fabricate bioelectrode interface, a sensitive electrochemiluminescence (ECL) biosensor was constructed for Gal T activity analysis based on the recognition between artocarpus integrifolia lectin (AIA) and galactose, integrating with a dual signal amplification strategy from the xanthine oxidase (XOD) and AIA multi-labeled gold nanorod nanoprobes. The gold nanorods promoted the electron transfer on the electrode interface and were also employed as carriers of AIA and XOD due to their large surface area. Furthermore, both the gold nanorods and XOD catalyzed the ECL reaction, which dramatically amplified the ECL signal of luminol in the presence of hypoxanthine (HA) and oxygen. The as-proposed ECL biosensor exhibited high sensitivity on the detection of Gal T activity and a detection limit of 9 x 10(-4) U mL(-1) was obtained. This assay was successfully applied for the analysis of Gal T activity expression in different cell-lines and inhibition detection, showing great potential for glycosyltransferase activity analysis and inhibitors screening in clinic diagnostics. (C) 2016 Elsevier B.V. All rights reserved.