In this paper, we rationally design a novel G-quadruplex-selective luminescent iridium (III) complex for rapid detection of oligonucleotide and VEGF165 in microfluidics. This new probe is applied as a convenient biosensor for label-free quantitative analysis of VEGF165 protein from cell metabolism, as well as for studying the kinetics of the aptamer-protein interaction combination with a microfluidic platform. As a result, we have successfully established a quantitative analysis of VEGF165 from cell metabolism. Furthermore, based on the principles of hydrodynamic focusing and diffusive mixing, different transient states during kinetics process were monitored and recorded. Thus, the combination of microfluidic technique and G-quadruplex luminescent probe will be potentially applied in the studies of intramolecular interactions and molecule recognition in the future.