The objective of this study is to develop a drug carrier to overcome the inherent drawbacks of 5-Fluorouracil (5-Fu), including low bioavailability, short half-life, and systemic toxicity. In the present work, mesoporous silica nanoparticles (MSNs) capped by chitosan (CS) to encapsulate 5-Fu (5-Fu MSNs/CS) were fabricated by sol-gel process, ultrasonic impregnation and emulsion cross-linking. The 5-Fu MSNs/CS microspheres exhibit pH-responsive drug release and remarkable drug encapsulation capacity, as well as perfect sphericity, high specific surface area (680.62 cm2/g), and uniform particle size (2.64 ± 0.05 um). The drug-loading content and encapsulation efficiency are 14.12 ± 0.53% and 82.21 ± 2.13%, respectively. The cumulative release of 5-Fu from MSNs/CS microspheres is fast and sustained at pH 5.0 (89.56 ± 0.97%) compared to that at pH 7.4 (57.88 ± 0.91%) in 96 h, and it is Fickian diffusion controlled. In conclusion, the MSNs/CS microspheres prepared in this study could be potential carriers for 5-Fu delivery.