Upconversion nanoparticles (UCNPs) have been proposed as a promising new class of biological luminescent labels because of their weak auto-fluorescence background, strong penetration ability under near-infrared (NIR) radiation, resistance to photobleaching, and low toxicity. Although UCNPs hold great promise in nanotechnology and nanomedicine, their applications in ECL fields still remain unexplored. Herein, a label-free, ultra-sensitive and selective electrochemiluminescence (ECL) assay is developed for detection of cyclin A2 by using highly efficient ECL graphene-upconversion hybrid. Being an important member of the cyclin family, cyclin A2 is involved in the initiation of DNA replication, transcription and cell cycle regulation through the association of cyclin-dependent kinases (CDK). Cyclin A2 is a prognostic indicator in early-stage cancers and a target for treatment of different types of cancers. However, the expression level of cyclin A2 is quite low, direct detection of cyclin A2 in crude cancer cell extracts is challenging and important for both clinical diagnosis of cancer in the early stage and the treatment. By chemically grafting cyclin A2 detection specific probe, a PEGlyted hexapeptide, to graphene-upconversion hybrid, the constructed ECL biosensor displays a superior performance for cyclin A2, which can not only detect cyclin A2 directly in cancer cell extracts, but also discriminate between normal cells and cancer cells. More importantly, the ECL biosensor has different responses between clinical used anticancer drug-treated and non-treated cancer cells, which demonstrates that the sensor can be potentially used for drug screening, and for evaluation of therapeutic treatments in early-stage cancers.