<p>Background: Acute Promyelocytic Leukemia (APL) is a subclass of acute myeloid leukemia. The chromosomal aberration in 95% of APL cases is t(15; 17) (q22; q21), which prevents cell differentiation. Characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. The goal of this study was to develop a new and powerful Flow- FISH technique to detect the long isoform (L) of PML-RARa fusion transcript in NB4 cell line.<br /> Methods: To achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% Triton X-100 and 0.2% saponin were used for the permeabilization step .In hybridization, a wide range of times and temperatures were used and probe was designed in FRET system. Results were confirmed by fluorescent microscope assay and reverse transcription PCR.<br /> Results: In the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of PML-RARa transcript. Using standard fixation and permeabilization protocol of 2% PFA and 0.2% saponin gave the best fluorescent results in flow cytometry. Also, results indicated that the optimum time and temperature for hybridization was 2 <em>hr</em> at 42^o<em>C</em>. The results of reverse transcription PCR and fluorescent microscopy confirmed the presence of PML-RARa transcript.<br /> Conclusion: The concordance between the results of Flow-FISH and those of two other techniques including reverse transcription PCR and FISH indicated that this method would be applicable as a diagnostic test for APL in clinical samples and MRD monitoring.</p>