Objective To systematically evaluate the physicochemical properties, biosafety and collagen regeneration effect of a novel poly-L-lactic acid (PLLA) microsphere filler, and to conduct a comparative analysis with a commercially available comparable product. Methods The physicochemical properties of the novel PLLA microsphere filler were analyzed by scanning electron microscopy, infrared spectroscopy and particle size analyzer. According to GB/T 16886.5-2017 standard, MTT assay was used to test its cytotoxicity. The animal model of rabbit dermal injection was constructed, and the experimental group (novel PLLA microsphere filler), positive control group (commercially available comparable product) and blank control group (physiological saline) were set up. The biological safety and collagen regeneration efficiency of the material were comprehensively evaluated by gross observation, pathological staining and image analysis. Results The microspheres of this novel filler exhibited regular morphology, smooth surface and uniform dispersion, with an average particle size of 42.31 μm and narrow particle size distribution (D50=39.75 μm, D90 =72.50 μm, D10 =14.91 μm). Its chemical structure was consistent with the characteristics of poly-L-lactic acid. The relative cell viability was 113.20%, with toxicity grade 0 and no cytotoxicity. Animal experiments showed that the experimental group presented slighter local swelling at injection sites,with shorter regression time, which basically returned to normal within 3 days. The inflammatory response in the experimental group was mild, with only a small amount of lymphocyte infiltration, no infiltration of neutrophils or eosinophils, and no adverse reactions such as tissue necrosis, granuloma, erythema or nodule formation. The collagen fiber content in the experimental group increased continuously over time, and the effect could last for more than 12 months. The collagen fiber area percentages at 1, 26, and 52 weeks after injection were 45.32%, 68.75%, and 62.38% in the experimental group, compared to 40.18%, 59.32%, and 51.24% in the positive control group, respectively. The percentage of collagen fibers at each time point (1, 26, 52 w) was higher in the experimental group than that in the positive control group. At the 130?week long?term follow?up, the materials in both groups had completely degraded, while the collagen retention level in the experimental group was higher than that in the positive control group. Conclusion The novel PLLA microsphere filler possesses favorable biocompatibility and safety, and can durably stimulate collagen regeneration, which demonstrates promising clinical application value.