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Abstract Background Microarray techniques have revolutionized genomic research by making it possible to monitor the expression of thousands of genes in parallel. As the amount of microarray data being produced is increasing at an exponential rate, there is a great demand for efficient and effective expression data analysis tools. Comparison of gene expression profiles of patients against those of normal counterpart people will enhance our understanding of a disease and identify leads for therapeutic intervention. Results In this paper, we propose an innovative approach, fuzzy membership test (FM-test), based on fuzzy set theory to identify disease associated genes from microarray gene expression profiles. A new concept of FM d-value is defined to quantify the divergence of two sets of values. We further analyze the asymptotic property of FM-test, and then establish the relationship between FM d-value and p-value. We applied FM-test to a diabetes expression dataset and a lung cancer expression dataset, respectively. Within the 10 significant genes identified in diabetes dataset, six of them have been confirmed to be associated with diabetes in the literature and one has been suggested by other researchers. Within the 10 significantly overexpressed genes identified in lung cancer data, most (eight) of them have been confirmed by the literatures which are related to the lung cancer. Conclusion Our experiments on synthetic datasets show that FM-test is effective and robust. The results in diabetes and lung cancer datasets validated the effectiveness of FM-test. FM-test is implemented as a Web-based application and is available for free at http://database.cs.wayne.edu/bioinformatics.