Abstract: Objective To study the preparation of oligonucleotide microarray for detecting vaccinia virus. Methods Oligonucleotide probes were designed and synthesized according to the specific genes of vaccinia virus. Sample DNA of the virus and the negative control sample were obtained and labeled by restriction display technique, followed by hybridization to the oligonucleotide microarray and scanned by Agilent scanner. Results Strong hybridization signals were detected from the viral DNA hybridized with the microarray, but were absent in the negative sample when positive probes were not used. Conclusion Distinct differences in the hybridization signals between the virus sample and negative sample and between the samples obtained in different phase of infection demonstrate high specificity and sensitivity of the microarray for vaccinia virus detection.