Presence of food allergens in various consumable products is a serious concern for the public health, especially for the infants. Therefore, real time quantification of the allergens in various food products has become one of the primary quests in food industries. In this contribution, we delineate fluorescence based quantification of a milk allergen (β-lactoglobulin, β-LG) in various dairy samples using a fluorogenic dansylated derivative of gemcitabine (GEM-DNS). The probe GEM-DNS is faintly emissive in aqueous solution but demonstrates a phenomenal escalation (more than 300 folds) in its fluorescence intensity under the influence of β-LG, due to the formation of GEM-DNS@β-LG complex. The decrease in torsional flexibility and hence, the TICT state formation for the probe in the GEM-DNS@β-LG complex significantly impedes the non-emissive decay pathways for the bound dye, triggering such enhancement in its fluorescence intensity. Moreover, this turn-on response of the probe is observed to be highly selective towards β-LG, enabling us to sense β-LG with limit of detection down to (1.18 ± 0.03) μM and (0.37 ± 0.01) μM in 20 % milk matrix and whey samples respectively. Finally, the estimated concentrations of β-LG have been found to be in nice compliance with the obtained concentrations from densitometric method, commonly used for protein determination. Thus, GEM-DNS provides a promising platform for β-LG quantification in several dairy and milk products using fluorescence based method, which can be used as a substitute for the conventional and complex methods used for β-LG quantification till date.