Synthetic colloid motors are the micro-/nanomachines which can harvest different types of energies into mechanical motion in the fluids. Since 2012, it has been one of important strategies for controlled fabrication of colloidal motors to combine effectively controlled molecular assembly (“bottom up”) with top-down approaches. Colloid motors based on controllable molecular assembly possess the advantages of: (1) mass production, (2) response to the external stimuli, and (3) access to multifunctionality. This review will summarize the progress on the controlled preparation, motion control, and biomedical applications of colloid motors through integrating multifunctional building blocks into assemblies. This review will also introduce the controlled preparation of bubble-propelled colloid motors based on layer-by-layer assembled microcapsules, nanotubes, shells, and two-dimensional plates, the fabrication of near-infrared-light-driven colloidal motors based on polyelectrolytic multilayer microcapsules and nanotubes, the controlled fabrication of biointerfacing colloidal motors, the effective control over the movement speeds, directions, and states of motors through integrating functional components, and the applications of motors in biomedical fields including drug targeted delivery, photothermal therapy, and biodetoxification.