A multi-probe fiber-optic evanescent wave biosensor has been developed based on the principle of evanescent wave while light propagates along optical fiber. A laser diode at 635 nm wavelength is used as the light source. The biosensor can interrogate five individual fibers at one time. Thus assays for five separate analytes can be conducted on the same sample. For all the five fibers, sensitivity limit of 0.01 nmol/L is obtained from the detection of Cy5 serial solutions with various concentrations. The response curves of five fibers have good consistency, the relative standard deviation is less than 10%. All the results are comparable with those that are obtained by a commercial biochip scanner. Real-time fluorescence of human IgG binding to goat anti-body IgG antibody immobilized on fiber core has been successfully detected.