A β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from hepatic extracts of Sotalia fluviatilis, order Cetacea. The protein was purified by using ammonium sulfate fractionation and four subsequent chromatographies (Biogel A 1.5 m, Chitin, Deae-Biogel and hydroxyapatite resins). After these purification steps, the enzyme was purified 380.5-fold with an 8.4% yield. The molecular mass (10 kDa) was estimated by SDS–PAGE and MALDI-TOF analysis. A Km of 2.72 mM and Vmax 9.5 × 10−6 μmol/(min.mg) were found for this enzyme, determined by p-nitrophenyl-β-d-hexosaminide substrate digestion. Optimal pH and temperature for β-N-Acetylhexosaminidase activity were 5.0 and 60 °C, respectively. Enzyme activity was inhibited by sodium selenate (Na2SeO4), mercuric chloride (HgCl2) and sodium dodecyl sulfate (C12H25SO4Na), and activated by zinc, calcium, barium and lithium ions. Characterization of the β-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.