The skin ulcer syndrome is a serious disease causing massive mortality and economic losses in cultured sea cucumber (Apostichopus japonicus). The causative agent was identified as Vibrio splendidus. As V. splendidus DNA was gained, the 16s-23s rDNA intergenic spacers were amplified by PCR, and cloned into pMD19-T vectors and finally sequenced. A fragment (about 177bp) was obtained by PCR, and it was labeled with digoxigenin-dNTP to form a dot-blot hybridization probe for V. splendidus DNA. The results showed that the probe was only positive for V. splendidus DNA, while it was all negative for DNAs of bacteria V. fluvialis, V. anguillarum, V. alginolyticus, V. parahaemolyticus, V. harveyi, V. vulnificus and Aeromonas hydrophila. The sensitivity test illustrated that the detection limit of the dot-blot hybridization probe was 6.25pg V. splendidus DNA. In addition, the probe was considered to be accurate in the detection of V. splendidus as the positive ratio was as high as 100% for the total samples from Qingdao, Weihai and Yantai areas. This probe was the first time to be established and applied in China to detect V. splendidus in A. japonicus. As it is sensitive and accurate in detection of V. splendidus, it would be helpful in the disease control and health management in cultured A. japonicus.