Fluorogenic substrates for chymosin [Dns‐Leu‐Ser‐Phe‐Trp‐Ala‐Leu‐OCH2Py (I), Dns‐Leu‐Ser‐Phe‐Met‐Trp‐Leu‐OCH2Py (II), Dns‐Leu‐Ser‐Leu‐Trp‐Ala‐Leu‐OCH2Py (III), Dns‐Leu‐Ala‐Phe‐Trp‐Ala‐Leu‐OCH2Py (IV), Dns‐Leu‐Ser‐Phe‐Leu‐Ala‐Leu‐OCH2Py (V) and Dns‐Leu‐Ser‐Phe‐Phe‐Ala‐Leu‐OCH2Py (VI)] were synthesized by a solution method. The obtained substrates I‐VI were cleaved specifically (between the Phe and Trp residues for substrates I and IV, the Phe and Met residues for substrate II, the Leu and Trp residues for substrate III, the Phe and Leu residues for substrate V, and the Phe and Phe residues for substrate VI) by chymosin. The fluorescence of substrates I‐IV (345 nm) increased with their hydrolysis, and hydrolysis rates were obtained by measuring the increase in fluorescence. The minimum detectable chymosin concentrations for substrates I and IV were about 1 nM; those for substrates II and III were about 4 and 2 nM. This assay method is very sensitive, and it is possible to determine the chymosin activity rapidly and easily. Substrates I and IV‐VI were hydrolyzed by chymosin two times faster than substrates II and III. The effect of the am