Lipopolysaccharide (LPS) as the component of cell membrane on gram-negative bacteria played a central role on inflammatory inducer to stimulate a multi-system host response. Herein, a surface-enhanced fluorescent (SEF) sensor was developed for LPS analysis based on shell-isolated nanoparticles (SHINs). The fluorescent signal of CdTe quantum dots (QDs) was amplified by silica shell-coated Au nanoparticles (Au NPs). The 3D finite-difference time-domain (3D-FDTD) simulation revealed that this enhancement was due to local electric field amplification. This method has a linear detection range of 0.1-20 μg/mL and a detection limit of 64 ng/mL for LPS. Furthermore, the developed method was successfully applied for LPS analysis in milk and human serum sample. The results indicated that the as-prepared sensor has significant potential for selective detection of LPS in biomedical diagnosis and food safety.