To study the viral etiology of acute conjunctivitis broke out in Shenzhen in 1997.Isolated the virus from eye and pharynx swabs of acute conjunctivitis in acute stage, and detected virus nucleic acid by polymerase chain reaction (PCR). DNA of viral strains were subjected to restriction enzyme analysis using several endonucleases. Also, neutralizing antibody of paired sera were detected.The positive virus isolation rates of 14 pharynx and eye swabs were 71.43% and 64.29%, respectively; and that of the virus DNA by PCR were 78.57% and 71.43%, respectively. All of the strains isolated were adenovirus type 3. Three paired viral strains chosen and subjected to restriction enzyme analysis proved belonging to Ad3a2 gene type. The neutralization test of paired sera of 24 cases showed that the antibody titers had 4 fold rising or above. Neutralization test of isolated viruses with paired sera of itself was carried out, the level of neutralizing antibody in convalescent stage was above 4 times as compared with that of acute stage.The results showed that the outbreak of acute conjunctivitis occurred in 1997 in Shenzhen was caused by adenovirus type 3.