The activity of α-l-fucosidase (FSC) has been measured for the first time in Fiore Sardo PDO (Protected Designation of Origin) raw milk cheese. To do this, a RP-HPLC method has been developed, validated and tested on a reliable sampling of cheese experimentally produced in laboratory batches. Three experimental factors have been considered in this work: the thermal treatment undergone by the milk, the lactation period and the ripening time of cheese. Results obtained have evidenced: (i) a meaningful reduction in the activity of FSC from cheeses produced using raw milk to those obtained by thermized milk; (ii) an increase in the activity of FSC during the first months of lactation period (from December to February), followed by a substantial constancy in the central and final months of lactation (from February to May); (iii) the enzyme activity is independent of the ripening time. This method might be useful in revealing frauds related to the use of mild thermal treatments of the milk when this is not allowed as for Fiore Sardo PDO cheese but also for several PDO cheeses for which the specifications establish that raw milk only must be used for their production.