Kaempferol is well known for the desirable properties in radical scavenging, oxidation resistance and inflammation resistance. However, the clinical translation of kaempferol for inflammatory disease treatment is still restricted by the poor solubility, undesirable stability and dissatisfied bioavailability. In this manuscript, multi-responsive kaempferol loaded fibroin nanoparticles (KF-NPs) were fabricated to overcome these shortages in biomedical application. The experimental results suggested that the negative charged KF-NPs (−25.2 ± 0.3 mV) had favorable average particle size (151 ± 1.6 nm) with uniform distribution. Noticeably, the accumulative drug release of fibroin nanoparticles dramatically increased in environment with acidity, glutathione (GSH) or reactive oxygen species (ROS), indicating the on-demand intracellular drug release properties. In vitro experiments revealed that the KF-NPs were biocompatible and can be internalized by cells. Moreover, KF-NPs had synergistic effect in downregulating the expression of TNF-α and eliminating the intracellular reactive oxygen. These results suggested that KF-NPs were promising therapeutic platform for anti-inflammatory treatments.