The scarcity of ATP-binding cassette subfamily B member 1 (ABCB1, also known as P-glycoprotein, P-gp) inhibitors suitable for clinical application in improving multidrug resistance (MDR) promotes the development of drugs aimed at reversing MDR. In this work, we reported a comprehensive study for the first time about the reversal activity of β-carboline derivatives on ABCB1-mediated MDR. Among 48 synthesized derivatives, compound K27 significantly increased the sensitivity of ABCB1-mediated MDR SW620/AD300 cells to paclitaxel (PTX) (IC