A point-of-care testing platform, consisting of smartphone, miniature electrochemical workstation, and screen-printed carbon electrode (SPCE) modified by gold nanoparticle (AuNPs) and multi-walled carbon nanotubes (MWCNTs), is fabricated for the ultrasensitive detection of paclitaxel (PTX) in human serum and injection solution. To enhance conductivity of the sensing system, MWCNTs concentration and AuNPs electrodeposition time were optimized. The AuNPs/MWCNTs effectively increase the working electrode area of SPCE by a factor of 1.46, contributing to improved electrochemical performance. The steps of electrode surface modification and the characterization of AuNPs/MWCNTs/SPCE were investigated by differential pulse voltammetry, impedance spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy. The sensor shows good linearity between current response and PTX concentration in 0.2 M phosphate buffer at pH = 7.4 (0.05-10 μM, with a limit of detection (LOD) of 1.7 nM) and human serum (0.5-30 μM and a LOD of 3.6 nM). The recoveries range from 89.91 to 103.36% and 91.42 to 103.73% in human serum and injection solution, respectively, with satisfactory relative standard deviation. Moreover, the sensor has excellent stability during 8 weeks and exhibits outstanding specificity and reproducibility towards PTX detection, providing a possible option for PTX determination in practical application such as therapeutic drug monitoring and drug quality control.