An ultrasensitive disposable immunosensor based on a gold nanoparticles (AuNPs)/indium tin oxide (ITO) substrate has been constructed to detect ketamine (KET) conveniently. In this study, we obtained a AuNPs/ITO basic electrode by only a two-step ecofriendly drip-coating procedure. The self-assembled monolayers of AuNPs were used as a hetero-bifunctional cross-linker and activator, respectively. Ketamine antibody was used as a biorecognition determinant and immobilized onto the AuNPs/ITO through electrostatic interaction or Au−S or Au−N bonds. The architectures of AuNPs/ITO were characterized by transmission electron microscopy, scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy mainly. Electrochemiluminescence (ECL) was used to monitor the specific immune response between the antigen and antibody by measuring changes of intensity in the process of ECL. When the immune complex was incubated on the electrode, it blocked the electron communication and mass transfer, and the ECL signal was quenched. According to the quenched ECL signal, the quantification of KET can be realized. Under optimal conditions, the immunoassay showed a linear range from 10 to 100 pg/g. The low detection limit is 5.73 pg/g. The sensor has been applied to detect ketamine in human hair successfully. In addition, the immunosensor has excellent selectivity, outstanding reproducibility, and acceptable stability. What's more, the immunosensor exhibits low-cost, simplicity, small sample consumption, and easy miniaturization, which allows it to be integrated into portable platforms to be utilized on the site of crime easily.