The shape and function of micro-/nano-particles for loading drugs play an important role in the transportation process of the drug delivery system. This study successfully fabricated red blood cell (RBC)-mimicking Janus microparticles (JMPs) using doubleneedle electrospray, which contained porous Fe 3 O 4 magnetic nanoparticles (MNPs) and upconversion (UC) luminescent nanoparticles NaYF 4 :Yb 3+ ,Er 3+ and exhibited a unique two-compartment structure. The JMPs possessed excellent magnetic and UC luminescent properties, as well as good hemocompatibility and cellular compatibility. Importantly, the two compartments of the Janus structure significantly enhanced the UC luminescent intensity compared to single compartment microparticles by reducing direct interactions between Fe 3 O 4 MNPs and UC luminescent materials. By utilizing the porous structure of Fe 3 O 4 MNPs, doxorubicin hydrochloride was loaded into Fe 3 O 4 MNPs and then embedded into the JMPs resulting in a slow-release effect. Given the excellent properties of magnetic and UC luminescence, biocompatibility, and drug slow-release ability, the RBC-mimicking JMPs have significant potential for drug delivery and bioimaging applications.