Rapid diagnosis and early treatment of invasive aspergillosis is crucial for the management of the patients with haematological malignancy. We evaluated 358 sera from 78 febrile neutropenic episodes in patient with invasive aspergillosis (IA) (one proven, 17 probable, and 60 possible) and 83 episodes in patients with no IA according to the EORTC/MSG criteria. Patient's specimens were tested by Mycassay Aspergillus PCR (first commercial real-time PCR test) and in house real-time PCR to investigate the presence of Aspergillus DNA, and by ELISA for detect the galactomannan (GM) antigen. We systematically investigated the medical background that can be effective on the test results. The hospitalisation period was longer in proven/probable episodes when compared with no IA (P = 0.001) and possible episodes. With regard to duration of neutropenia, the differences between both proven/probable with no IA (P = 0.023) and possible with no IA (P = 0.002) were highly significant. Similarly, the rates of T cell suppressant therapy in group proven/probable and possible episodes were significantly higher than in no IA (P = 0.005). There are significant differences in the performance of GM and PCR-based tests among studies, and standardisation is required. Therefore, it can be useful to determine the effective factors on these tests. The use of larger volume of sera improved the performance of real-time PCR for detection of Aspergillus DNA in high-risk adult patients in the present study. Some host factors such as duration of neutropenia and administration of T cell suppressants related to the development of IA.