We present a room temperature nonlinear mass sensing based on a hybrid spin-nanoresonator system with the microwave pump–probe technique and the spin readout technique, which includes a single spin of nitrogen–vacancy (NV) center in diamond and a nanomechanical cantilever. The resonance frequency of the nanoresonator can be measured with the nolinear Kerr spectrum, and the parameters that influence the nolinear Kerr spectrum are also investigated. Further, according to the relationship between frequency shifts and variable mass attached on the nanoresonator, this system can also be used to detect the mass of DNA molecules with the nolinear Kerr spectrum. Benefiting from the single spin of the NV center in diamond has a long coherence time at 300 K, the hybrid system can realize room temperature mass sensor, and the mass response rate can reach 2600 zg/Hz.