An ultrasensitive and turn off-on electrochemiluminescence (ECL) sensing platform was constructed by using manganese dioxide carboxylated graphitic carbon nitride (MnO2/C-g-C3N4) nanocomposite as the probe for L-cysteine (CySH) detection. In this work, we synthesized the MnO2/C-g-C3N4 tags via a facile one-step method. The ECL of C-g-C3N4 nanosheet can be quenched by the MnO2 nanosheet due to electrochemiluminescence energy transfer (ECL-ET) from C-g-C3N4 to MnO2, C-g-C3N4 and MnO2 as the energy donor and energy acceptor, respectively. However, the addition of CySH would lead to the elimination of ECL-ET, resulting in sufficient recovery of ECL intensity because it reduced MnO2 to Mn2+. Under the optimized conditions, the fabricated biosensor exhibited a wide response to CySH in the range of 0.50 nM to 1.0 mu M and a low detection limit of 0.18 nM. Moreover, this convenient sensing system can successfully detect CySH in real human urine samples with satisfactory recoveries, which could provide a new way to detect CySH quickly and sensitively. (C) 2019 The Electrochemical Society.