Abstract: Novel β-mannanase resources will be explored and characterized to establish a robust enzymatic foundation for the efficient degradation of mannan-rich substrates. A β-mannanase-producing fungus was isolated from soil samples and identified via morphological observation and internal transcribed spacer sequence analysis. The liquid fermentation conditions were optimized through a combination of single-factor experiments, complex carbon source screening, and response surface methodology. The target enzyme was purified to homogeneity using ammonium sulfate fractionation followed by strong anion-exchange chromatography. Subsequently, its enzymatic properties, substrate specificity, and hydrolysis patterns were systematically investigated. A high-yield strain, Purpureocillium lilacinum CF309, was identified. The optimal medium consisted of 6.5% complex carbon source (konjac flour to palm kernel cake, 1:5, m/m), 2.5% tryptone, and 1.3% Triton X-100. Under optimized conditions (30°C, initial pH 6.0, 4 d), the β-mannanase activity reached 640.4 U/mL. The purified enzyme, designated as PlManA, exhibited an apparent molecular mass of 35.0 kDa and a specific activity of 365.7 U/mg, with a 4.1-fold purification and an 8.4% yield. PlManA exhibited an optimal pH of 6.0 and maintained high stability within a broad pH range (5.0–9.0). The optimal temperature was 65°C, with over 70% residual activity retained after incubation at 50°C for 30 min. PlManA demonstrated high catalytic efficiency towards locust bean gum and konjac flour, primarily yielding mannooligosaccharides with a degree of polymerization of 2–6 and higher. The β-mannanase PlManA from P. lilacinum CF309 possesses good pH tolerance and favorable thermostability. Its distinct hydrolysis profile highlights its significant potential as a biocatalyst for applications in the food industry.