An aptamer-based "sandwich" approach combined with the chemiluminescence (CL) analysis was developed for the capture and detection of rare cells on a microfluidic chip. Aptamers were immobilized on microfluidic channels to achieve capture and isolation of the specific cells from a cell mixture. The capture efficiency for target cells was more than 70% with the purity greater than 97%, when the content of the target cells was between 0.5% and 10% in the initial cell mixture. Gold nanoparticles (Au NPs) modified with aptamers were then added in to bind on the cells and trigger a CL reaction. A satisfactory linearity of the log/log calibration curve between the CL intensity and the number of target cells was observed with a low detection limit of 30 target cells in a 3 μL cell mixture. Spiked whole blood samples were also used to verify the practicality of the present method. This work demonstrated the potential application of the cheap and rapid CL detection into the early diagnosis of cancers.