This study describes the preparation, characterization, and biosensing applications of Radio Frequency (RF) plasma-modified magnetic micro/M/Janus micromotors. In the first part of the study, two different Janus micromotors were fabricated. Firstly, Fe3O4 magnetic micro/particles were functionalized with –OH groups under plasma treatment, and then Fe3O4 and Fe3O4-OH NPs/Platinum (Pt) (Fe3O4/Pt and Fe3O4-OH/Pt) micromotors were prepared by Pt coating of one side of Fe3O4 and Fe3O4-OH nanoparticles using by RF magnetron sputtering method. The work is the first example of RF rotating plasma preparation of modified magnetic nano/particle-based motors. Thus, it is of great interest to nano/micromachinery field. The synthesized micromotors were characterized by scanning electron microscopy (SEM) and energy-dispersive X-Ray spectroscopy (EDX) analysis. The dependence of the mobility of the motors on fuel concentration was evaluated. High speeds of 255 μm s− 1 and 128 μm s− 1 at 5% hydrogen peroxide (H2O2) were observed for Fe3O4/Pt and Fe3O4-OH/Pt micromotors, respectively. Besides these remarkable values, long lifetimes of 15 min with Fe3O4/Pt micromotors and 20 min with Fe3O4-OH/Pt micromotors were achieved. In the second part of the study, these Janus micromotors were used in miRNA-21 biosensing. The changes in the fluorescence intensity and in the speed of micromotors were examined after hybridization. Performances of these two novel micromotors were compared to present their potential use in early cancer diagnosis. Promising results with the functionalized Fe3O4-OH/Pt micromotors were obtained.