To detect drug concentration in tacrolimus solution, an anchor planar millifluidic microwave (APMM) biosensor is proposed. The millifluidic system integrated with the sensor enables accurate and efficient detection while eliminating interference caused by the fluidity of the tacrolimus sample. Different concentrations (10–500 ng mL⁻¹) of the tacrolimus analyte were introduced into the millifluidic channel, where it completely interacts with the radio frequency patch electromagnetic field, thereby effectively and sensitively modifying the resonant frequency and amplitude of the transmission coefficient. Experimental results indicate that the sensor has an extremely low limit of detection (LoD) of 0.12 pg mL⁻¹ and a frequency detection resolution (FDR) of 1.59 (MHz (ng mL⁻¹)). The greater the FDR and the lower the LoD, the more the feasibility of a label-free biosensing method. Regression analysis revealed a strong linear correlation (R² = 0.992) between the concentration of tacrolimus and the frequency difference of the two resonant peaks of APMM. In addition, the difference in the reflection coefficient between the two formants was measured and calculated, and a strong linear correlation (R² = 0.998) was found between the difference and tacrolimus concentration. Five measurements were performed on each individual sample of tacrolimus to validate the biosensor's high repeatability. Consequently, the proposed biosensor is a potential candidate for the early detection of tacrolimus drug concentration levels in organ transplant recipients. This study presents a simple method for constructing microwave biosensors with high sensitivity and rapid response.