Milrinone (MIL) is an essential cardiac inotrope drug for cardiovascular disease. In this study, a simple nanosensor based on a nanodiamond carbon paste electrode modified with flower-like ZnO particles was developed to determine MIL electrochemically rapidly and accurately. The flower-like ZnO nanoparticles were synthesized by a simple and cheap sonochemical method. The structure and morphology of the ZnO nanoparticles were investigated by Fourier transform infrared spectrometer and scanning electron microscopy. The electron transfer properties of bare carbon paste electrodes, nD@CPE, CPE/ZnO, and flower-like carbon paste electrodes modified with ZnO and decorated with nanodiamonds (nD@CPE/ZnO) were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). It was confirmed that the nanosensor, which was electrochemically characterized by EIS and CV, was designed to increase electrical conductivity and facilitate electron transfer upon the modification of CPE with nanomaterials. The electrochemical behavior of MIL on the modified electrode was investigated by differential pulse voltammetry (DPV). Owing to the developed nD@CPE/ZnO nanosensor, the peak current of 10 mu M MIL in pH 4.0 BR buffer solution was found to be approximately 19 times higher in DPV compared to bare CPE. Under optimum conditions, the constructed nanosensor offered a linear calibration curve of concentration ranging from 0.2 to 10 mu M with the detection limit (LOD) and quantification limit (LOQ) 41 nM and 136 nM, respectively, for MIL in pH 4.0 Britton Robinson buffer solution. The same electrode was used for stability control after 7 days and its current response was found to decrease to 96.7%. The developed method was successfully used for the determination of MIL in human serum and urine samples. The method based on the use of modified nD@CPE/ZnO, showed linearity from 0.6 to 10 mu M in serum and 0.8-10 mu M in urine. LOD values were found as 128 nM and 176 nM in serum and urine samples, respectively. According to these results, this optimized method showed reasonable sensitivity for the determination of MIL in serum and urine samples.