In this study, a three-phase laminar flow microfluidic chip (TPL chip) combined with HPLC was developed for monitoring free and total concentrations of paclitaxel (PTX) in blood simultaneously. A diluted whole blood sample (aqueous phase) was introduced into the chip, ethyl acetate (organic phase) was introduced into the chip for extraction, and an interphase was used to prevent the blood sample from coming into direct contact with the organic phase. Because only free drug can quantitatively diffuse into the organic extraction phase and the free drug fraction has a linear relationship with the dilution factor of blood, both the free and total drug concentrations can be obtained by detecting the concentration of paclitaxel in the organic extraction phase. The governing factor such as flow rate for extraction was optimized. Docetaxel was used as an internal standard. The reliability of the quantitative diffusion of molecules in the TPL chip was proved by the methodological investigation of PTX in PBS sample, which showed a good linearity in the concentration range of 0.5 - 100 µg/mL and a detection limit of 7 ng/mL. Good repeatibilities for retention time (RSD of PTX is 1.23%, docetaxel is 1.14%, n = 5) and peak area ratio of PTX to docetaxel (RSD is 4.38%) were obtained. For blood sample analysis, only 100 µL of sample was needed and whole pretreatment was finished in 35 min, and a recovery of 94~117% were obtained. The provided method showed advantages in fast analysis speed, minimum sample handing, and potential ability of automation, and integration. Copyright © 2020. Published by Elsevier B.V.