There were approximately 93,060 deaths from cancers in Poland in 2008, and about 105,000 are predicted for the year 2025. Early detection of cancer is a major problem throughout the world, which is why many researchers are still looking for specific and sensitive markers of malignant tumors. Our work is a review of recent publications on activity of N-acetyl-β-D-hexosaminidase (HEX) and its isoenzymes A (HEX A) and B (HEX B) as potential markers of malignant tumors. HEX is the most active of the lysosomal exoglycosidases, taking part in degradation of glycoconjugates (glycoproteins, glycolipids, proteoglycans). HEX cleaves N-acetyl-D-glucosamine and N-acetyl-D-galactosamine from non-reducing ends of oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans. The activity of HEX, and its isoenzymes A (HEX A) and B (HEX B), was determined by spectrophotometric and isoelectric focusing methods. There was a statistically significant increase in activity of HEX in tumors of the kidney, pancreas, thyroid, colon, ovary, brain, salivary gland, stomach and larynx, which suggests potential applicability of HEX and its isoenzymes in cancer diagnosis.