Rapid and efficient measurement of cancer cells is a major challenge in early cancer diagnosis. In the present study, a miniature multiplex chip was created for in situ detection of cancer cells by implementing a novel graphene oxide (GO)-based Förster resonance energy transfer (FRET) biosensor strategy, i.e. assaying the cell-induced fluorescence recovery from the dye-labeled aptamer/graphene oxide complex. Fluorescence intensity measurement and image analyses demonstrated that this microfluidic biosensing method exhibited rapid, selective and sensitive fluorescence responses to the quantities of the target cancer cells, CCRF-CEM cells. Seven different cancer cell samples can be measured at the same time in such a microfluidic chip. The linear response for target CCRF-CEM cells in a concentration range from 2.5 × 10(1) to 2.5 × 10(4) cells mL(-1) was obtained, with a detection limit about 25 cells mL(-1), which is about ten times lower than those of normal biosensors. The novel fluorescence biosensing microfluidic chip supplies a rapid, visible and high-throughput approach for early cancer diagnosis with high sensitivity and specificity.