This study was aimed to establish the method of quantitative PCR (q-PCR) of fungi in peripheral blood for diagnosis of invasive fungal infections in patients with hematologic malignancies, and to preliminarily assess the diagnostic value of this method. The 18S rDNA-ITS1 area of high consensus sequence of fungi was selected to design primer and probe, the DNA of fungal species was extracted and q-PCR was performed to evaluate the sensitivity and specificity of the primer and probe. The standard product of fungal DNA was prepared by using pGEM-T plasmid and the fungal DNA in blood of patients was quantitatively detected. The results showed that the positive was found in 12 Aspergillus and 14 Candida species according to q-PCR detection, while there was no significant difference of fungal distribution between plasma, mononuclear cells and leukocytes (p<0.05). Receiver-operating characteristic analysis of q-PCR showed that the cut-off value for clinical diagnosis of invasive fungal infection was 8 copies/ml whole blood, its sensitivity, specificity, positive and negative predictive value and kappa were 0.84, 0.9, 0.955, 0.692 and 0.679 respectively. It is concluded that the fungal q-PCR assay may be used as an early diagnostic method for invasive fungal infections in patients with hematologic malignancies.