Introduction: Characterization of the relative electron density (𝜌e) of the body tissues is routinely provided by scanning the commercial phantom like RMI ۴۶۷ at ۱۲۰ kilovoltage (kVp) in radiotherapy planning. Recent studies showed that the calibrating Hounsfield Unit– 𝜌e curve could be obtained linearly using dual energy computed tomography (DECT) algorithm in commercial phantom like RMI ۴۶۷. The aim of this study was to produce a more accurate calibrating HU–𝜌e curve by constructing an in- house phantom and applying dual energy algorithm. Materials and Methods: An in-house water filled phantom (۳۳cm diameter) was made including ten water solutions plus composite cork as tissue substitute materials (TSM), and scanned at four kVps by multi-detector four slice CT. The dual energy algorithm was applied to two combination scans (۸۰-۱۴۰ and ۱۰۰-۱۴۰ kVp) and the linear HU–𝜌e curves were produced. The stoichiometric method reproduced the HUs of ۳۱ real body tissues, that their compositions are available in ICRU-۴۶ report. The HU–𝜌e curves for both kVp combination scans were produced. The t-test and compare means were performed between the mean and standard deviation of the relative and absolute differences (%) of the 𝜌e of ۳۱ ICRU real tissues calculated for ۱۲۰ kVp and both kVp combination scans in the current and previous studies, respectively Results: Applying an energy subtraction algorithm mitigated the 𝜌e calculation error of real tissues. The mean and standard deviation of the relative difference between the 𝜌e of ۳۱ ICRU tissues (–۰.۲۳±۱.۸۹) were statistically significant compared with the mean and standard deviation of the ۳۰ ICRU tissues (۰.۸۰±۱.۵۸), which extracted from the RMI ۴۶۷ phantom at ۱۲۰ kVp in previous study (p<۰.۰۲۴). The mean and standard deviation of absolute differences of the calculated 𝜌e of the ۳۱ ICRU tissues at ۱۰۰-۱۴۰ kVp combination scans (۰.۱۴±۰.۱۱) compared to previous study using ۱۰۰-۱۴۰kVp scan (۰.۳۰±۰.۴۰) in a second generation dual source CT, were statistically significant (P<۰.۰۳۵). Conclusion: The stoichiometric calibration method and closeness of the 𝜌e of ۱۱ TSMs can result in statistically significant smaller discrepancies in calculating the 𝜌e of real tissues at ۱۲۰ kVp, compared to the previous studies with RMI ۴۶۷. The stoichiometric fitting parameters were highly affected by beam hardening artifacts and image noise, especially at ۸۰ kVp. Applying the energy subtraction algorithm can offer further error mitigation in 𝜌e calculation of real tissues by spectral separation and reduction of beam hardening artifacts and noise in two kVp combination scans compared to previous studies. Therefore, a dual energy algorithm in combination with stoichiometry can be used to decrease errors in calculation of the 𝜌e of real tissues, and could be used for radiotherapy planning and material differentiation in clinical practice.