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Abstract Background Opioids abuse and related deaths are increasing in the world. Therefore, the design of new analytical methods for detection of opioids in biological samples is necessary for clinical and forensic settings. Methods In this study, dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography with photo diode array detector (HPLC-PDA), as a new and sensitive method were examined for the extraction and determination of morphine, codeine and methadone in postmortem urine samples. Effective factors on DLLME were optimized. The extracts were analyzed by HPLC-PDA using a Eurospher® C18 column (250 mm × 4.6 mm, particle size: 5 μm). Results The volumes of chloroform as the extraction solvent and acetone as the dispersive solvent were selected 300 μl and 500 μl, respectively. The optimum pH 9.8 and extraction time was 0.5 min were selected. Under optimum condition, the enrichment factor and the recovery of morphine, codeine, and methadone spiked into postmortem urine samples were in the range of 175–215.8 and 87.5–107.9%, respectively. Calibration curves for each analyte are linear in the range of 0.5–100 μg ml− 1. Limit of detection (LOD) for the analytes was in the range of 10–25 μg l− 1. Finally, the proposed method was successfully applied to 50 postmortem urine samples for determination of the opioids. Conclusions The proposed method is an easy, fast, low cost and efficient for the extraction and determination of opioids in postmortem urine samples and should be considered as analytical method for determination of opioids in forensic and clinical toxicology labs.