We report on thrombolysis using urokinase (UK)-coated Fe3O4 magnetic nanoparticles (MNPs) under an oscillating magnetic field in microfluidic channels under different conditions. With the help of a theoretical model and the multiphysics simulation software COMSOL, we analyzed the magnetic force and motion of the MNP aggregation in the oscillating magnetic field induced by an energized coil. On the basis of the steady flow theory, we analyzed the factors affecting the thrombolysis efficiency, such as the bending angle, the diameter of the microfluidic channel, the mass fraction of MNPs, and the strength of the magnetic field. Thrombolysis efficiency is considered an important factor in stroke therapy clinics because of strict time restrictions. The medium in a microfluidic channel will cause a rapid velocity with functionalized nanoparticle (NP) movement and rotation under an alternating field. Thus, an increased flow pressure will act on the target and accelerate the release of the enzyme or drug attached to the NPs to dissolve the thrombus chemically. In vitro experiments revealed that increases in the bending angle and diameter of the microfluidic channel result in a decrease in the efficiency of thrombolysis using UK-coated MNPs. The increase in the mass fraction of UK-coated MNPs results in a faster increase in thrombolysis efficiency than that of pure UK. The increase in the peak amplification of the oscillating magnetic field also improves the thrombolysis efficiency. The analysis of these factors can help sense the thrombolysis efficiency and enable doctors to make the right choices in clinical treatment.