The β-ketoadipate pathway is the major route for degradation of aromatic compounds by various soil microorganisms. Protocatechuate 3,4-dioxygenase, a key enzyme of this pathway and which is encoded by pcaGH genes, catalyses the ring cleavage of protocatechuate. Microorganisms harbouring pcaGH genes are widely distributed in the environment but little is known about their relative abundance within the total microflora. Hence, this paper reports the development of a real-time PCR assay to quantify the bacterial pcaH sequence that encodes the β sub-unit of the protocatechuate 3,4-dioxygenase. This real-time PCR assay was linear over seven orders of magnitude with a calculated efficiency of 99% and sensitive up to 102 copies of the pcaH sequence per assay. Real-time PCR analyses performed on six soils with different physico-chemical properties, revealed pcaH densities ranging from 103 to 104 copies of pcaH ng−1 of soil DNA, which corresponded to approximately 0.2–10.9% of the total bacterial community. The sequencing of real-time PCR amplicons yielded 48 deduced amino acid sequences that exhibited 44–100% identity to known bacterial PcaH sequences, thereby confirming the accuracy of this real-time PCR assay.