Silk fibroin presents a good advantage as a drug carrier for drug delivery, due to the excellent biocompatibility, biodegradability and tunable drug loading and release properties. In this work, we constructed silk spheres by phase separation of the regenerated silk fibroin (RSF) solutions with different MW and polyvinyl alcohol; and it was revealed that MW of RSF can affect the structure, size, surface potential and drug loading and release efficiency of silk spheres. Silk spheres prepared from high MW of RSF were found to load more macromolecular drug with negative charge compared to middle and low MW of RSF. However, for the positive charge and low MW drug, the silk spheres prepared from low MW of RSF could present a high loading efficiency compared to other carriers. Finally, a positive drug with low MW, streptomycin was encapsulated in silk spheres prepared from low MW of RSF, and displayed a long bactericidal and bacteriostatic effect compared to bared streptomycin solution. The results obtained provide guidelines for the modification and options of drug transport vehicles for more efficient drug delivery and utilization through a simple, rapidly constructed, applicable and low-cost drug carrier.