In this study nickel nanoclusters (NiNCs), was promised as novel and economic electrochemiluminescence (ECL) emitter for highly sensitive and selective determination of creatinine in the presence of molecularly imprinted polymer (MIP). The uniform magnetic graphene oxide (GO-Fe3O4) MIP film was established on the surface of ITO electrode and Ni NCs-embedded in MIP, showed a strong anodic ECL emission using tri-n-propylamine (TPrA) as coreactant. During the ECL process, TPrA was oxidized, and Ni NCs got the energy to generate excited state Ni NCs* for light emission. When the imprinted cavities were occupied by creatinine, the ECL emission of Ni NCs on the MIP-modified electrode surface was efficiently quenched. Under optimal conditions, the proposed sensor demonstrated ultrasensitive and accurate analytical performance toward creatinine detection with a linear range from 5 nM to 1 mM and detection limit of 0.5 nM (S/N = 3). The biosensor showed good specificity for creatinine determination compared to other compounds that having the chemical structure analogue or close to the template creatinine, when the concentration ratio of interference to creatinine was more than 100 times. Furthermore, the biosensor was successfully applied to determination of creatinine in human serum and urine samples with satisfactory results. So, this assay for creatinine detection possesses high sensitivity, good selectivity, excellent reproducibility and stability. We expect the combination of molecular imprinting system with ECL assay in the presence of Ni NCs as a new type of superior luminophore candidate can be developed for design of ultrasensitive sensors, biosensors and other measuring devices.