A highly sensitive and recyclable quartz crystal microbalance (QCM) biosensor was developed using chitosan (CS) and folic acid (FA), generating conjugates that are selectively recognized by MCF-7 cancer cell over-expressed folic acid receptors. The prepared CS-FA conjugate was characterized by UV-vis spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy and scanning electron microscopy further presented the morphology of the CS-FA conjugate interface. The hydrophilicity of films was characterized by measuring the contact angle. The recognition of MCF-7 cancer cells was investigated in situ using QCM. Captured by FA, the concentration of the MCF-7 cell was determined on-line using a quartz crystal microbalance and a wide linear range of 4.5 × 10(2) to 1.01 × 10(5) cells per mL was obtained, with a detection limit of 430 cells per mL. The fluorescence microscope further confirmed the specificity and biocompatibility of the constructed biosensor. In addition, the regeneration of the QCM biosensor was studied by using lysozyme. This receptor-bound ligand based QCM biosensor also showed good selectivity, and repeatability in the cell mixture. For the first time, this simple, economical and label-free chitosan-based QCM sensing was demonstrated, and such design could provide a promising detection strategy for sensitive detection of cancer cell over-expressed folic acid receptors.