Lectins are the tools for the determination of sugar chain structure. Recently, lectin arrays have become a popular new technology; therefore, lectins with specific sugar-binding properties are required. The objective of the study was to isolate a novel lectin from Pleurotus ferulae mushrooms and characterize its various biological activities. A novel lectin was extracted with deionized water, precipitated from the aqueous extract using 75% saturated (NH4)2SO4, and subjected on DEAE-cellulose followed by affinity chromatography on sepharose-6B. The activity was tested using hemagglutination assays, and carbohydrate-binding specificity was determined by glycan microarray analysis. Its effects on the mitogenic activity of mouse splenocytes were determined by MTT assay. The novel lectin was adsorbed on ion-exchange chromatography DEAE-cellulose and shown as a band with the molecular mass of 17.5 kDa on a SDS-PAGE and as a single 35.0-kDa peak in gel filtration on Superdex G-75. The hemagglutinating activity of the lectin was inhibited by D-glucose, lactose, D-galactose, and galactosamine. The lectin was stable on 60°C. The hemagglutinating activity of lectin was reduced by 50% at 70°C. At 80°C, it was further reduced to 6.25% of its original activity. The hemagglutinating activity was the highest at pH 6-9. Moreover, its hemagglutinating activity was inhibited by Mg2+ and Ca2+ ions. The lectin isolated from P. ferulae in the current study possessed highly potent hemagglutinating and proliferative activities toward mouse splenocytes.