The importance of fluorescence in situ hybridization (FISH) for pathological diagnosis has been increasing. However, the procedures utilized for a conventional FISH method with formalin-fixed paraffin-embedded tissue sections are complicated and it is difficult to perform as a routine laboratory test. In addition, there are difficulties with differentiation of targeted cells in observations with a fluorescence microscope. The present study reported a novel method that utilizes FISH in combination with fluorescence immunostaining as a simple double-detection technique that addresses these problems. Using this novel method, various genetic aberrations, as well as protein overexpression were easily visualized in isologous sections. In particular, FISH signals with our method clearly identify target cells in samples with poor differentiation between tumor cells coexisting with normal cells. It is proposed that this simple technique is widely applicable as a routine laboratory test and future developments are expected.