R454B is a relatively low Global Warming Potential refrigerant, and it has emerged as a promising substitute for traditional refrigerants. Understanding the heat transfer and fluid dynamics characteristics of R454B becomes important for applying it. An experimental setup was built for measuring heat transfer coefficient (HTC) and pressure gradient (dP/dz) in a 24-port microchannel tube. The test condition covers mass fluxes of 100–250 kg-m-2s−1, heat fluxes of 2–8 kW-m−2, and saturation temperatures of 10–30 °C. The mass flux had a significant impact on the HTC, particularly at higher vapor quality. Additionally, the heat flux also played a crucial role in determining the HTC, with a stronger influence observed at lower vapor quality. The saturation condition had a relatively minor effect on HTC, showing a positive impact at low vapor quality but a negative impact at moderate and high vapor quality. The dP/dz tended to increase with vapor quality. Both the mass flux and saturation condition exhibited strong effects on dP/dz. However, the heat flux had a negligible impact on the dP/dz. The study also compared the performance of R454B with R32, R1234yf, and three R32/R1234yf mixtures (85/15, 50/50, and 15/85 by mass). The results showed that R454B had a HTC close to R1234yf and the 85/15 mixture. However, it had the lowest dP/dz compared among the compared fluids. The results were compared to existing correlations and those with less error were recommended. This study pioneered the examination of R454B flow boiling in microchannel tubes, and employed Pearson correlation coefficient analysis which presents a first-of-its-kind quantitative analysis of the impact of operational conditions on HTC and dP/dz, marking a significant advancement in thermal systems optimization.