A biotin-conjugated photoactivatable phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was synthesized to identify sialidase. The free carboxylic group and N-acetyl substituent of sialic acid, which are important for recognition and enzymatic activity of sialidase, were conserved by the photolabeling compound as confirmed using analytical methods. The synthesized compound and DANA competitively inhibited starfish sialidase with a Ki value of 7.6 μM and 4.6 μM, respectively. Photo incorporation of the labeling compound to sialidase increased with irradiation time; 90% photo incorporation was achieved with more than 10-min irradiation, and labeling was completely inhibited by the addition of a competitive inhibitor. Starfish sialidase purified using high-performance gel filtration chromatography was subjected to photoaffinity labeling. A 50-kDa band was revealed to contain the sialidase active site by the photolabeling compound, and labeling was completely hindered in presence of the competitive inhibitor. Labeling specificity was ensured by the addition of the heat-deactivated standard protein chymotrypsinogen A to the reaction mixture. © 2008 Pharmaceutical Society of Japan.