The commercialization of lithium-sulfur (Li-S) batteries faces several bottlenecks, and the major two of which are the shuttle effect of polysulfides and the wild growth of Li dendrites, responsible for fast capacity decay and severe safety issues. As an essential component of Li-S batteries, the structure and properties of the separators are closely related to the above problems, and the exploration of multifunctional separators is highly sought-after. Herein, an integrated separator composited of defective graphene and polyimide (DG-PI) was innovatively fabricated by electrospinning combined with the laser-induced carbonization strategy. The all-in-one compact architecture with well-interconnected channels shows superior mechanical and thermal stability and wettability. More importantly, the PI nanofibers containing N–/O– functional groups can induce the uniform deposition of lithium on the anode surface, while the DG framework with abundant pentagonal/heptagonal rings and vacancies can strongly trap polysulfides and accelerate polysulfide transformation on the cathode side. The strong chemical interaction between the insulative PI layer and the conductive DG layer modulates the surface charge distribution of each other, leading to more prominent contributions to restraining lithium dendrites and shuttle effect. Therefore, the Li-S batteries based on the integrated DG-PI separators afford an excellent performance in protecting lithium anode (stable cycles of 200 h at 5 mA·cm⁻²) and good cycling stability with a low capacity decay of 0.05% per cycle after 700 cycles at 1 C. This work offers a new design concept of multifunctional Li-S battery separators and broadens the application scope of laser micro-nano fabrication technology.