In this experiment, a novel biotin-avidin conjugation probe was synthesized and employed in the detection of reverse-phase protein microarray. Firstly, the proportion of the biotin-avidin conjugation probe was optimized. Then the rat IgG and goat anti-rat IgG system was served as a model to optimize the fabrication conditions of reverse-phase protein microarray, including the non-specific absorption of streptavidin-Cy3 molecules, spotting buffer as well as protein activities. At last, the biotin-avidin conjugation probe was applied to the detection of the reverse-phase protein microarray. The results show that the protein microarray prepared by using BSA spotting buffer could prevent non-specific absorptions of fluorescent molecules and improve the sensitivity, effectively. In addition, compared with traditional biotin-avidin system, the detection limit could be improved four times using the biotin-avidin conjugation probe. In conclusion, the biotin-avidin conjugation probe has its merits of easy synthesis, low price and could be further conjugated with other signal amplification techniques, which is promising to be used in the detection of protein microarray.