The invention discloses a triplex seven-fold PCR detection primer set for virulence genes of Streptococcus agalactiae, including virulence genes sip, fbsA, hylB, cfb, sodA, DltR, cspA, ponA, bibA, srr-1, bca, PCR amplification primer pairs corresponding to iagA, scpB, fbsB, pavA, psaA, spb1, bac, cppA, lmb and cylE. The sequences of each primer are shown in the sequence table. The invention also discloses a reagent kit comprising the above primer set and a three-in-one seven-fold PCR detection method of the Streptococcus agalactiae virulence gene using the above primer set. The detection method uses sip, fbsA, hylB, cfb, sodA, DltR and cspA as the first group, ponA, bibA, srr-1, bca, iagA, scpB and fbsB as the second group, pavA, psaA, spb1, bac , cppA, lmb and cylE are the third group, and PCR reaction is carried out under the same reaction conditions at the same time, and the target sequences of 21 virulence gene target fragments are amplified at the same time. Source and typing of Streptococcus lactis host, and evaluate the variation of strain virulence gene profile.