This study aimed to establish an accurate and sensitive polymerase chain reaction (PCR) technique for the diagnosis of active human brucellosis in Egypt. We failed to extract Brucella DNA with a commercial kit, but an extraction kit designed in-house using 2 sets of primers [B4/B5 (223 bp) and JPF/JPR (193 bp)] was successful and more economical. The technique showed high sensitivity, specificity and accuracy. The PCR positivity increased significantly with increasing seropositivity titres by the standard tube agglutination test and showed 100% positivity in patients with positive blood cultures. We recommend using PCR as an alternative to culture for diagnosis of brucellosis.