The aim of the present study was to investigate impact of adding transglutaminase (TGase) under different conditions on making bio-yoghurt. The TGase was added before (B) and after (A) heat treatment of milk (92^oC/10min), followed by pre-incubation at 40^oC (B1, A1) or 50^oC (B2, A2) for 1h and inactivation at 80^oC/2min. Bio-yoghurt was prepared by inoculation the milk with yoghurt starter and B. infantis (1:1). Chemical properties of the bio-yoghurt were affected by both heat treatment of milk and pre-incubation temperatures of the enzyme. TGase mostly increased values of pH, NPN/TN and TVFA and decreased the acidity values. Viscosity and water holding capacity (WHC) were significantly increased when TGase was added, especially, after heat treatment of milk, followed by pre-incubation at 50^oC/1h (A2). The highest WHC values were correlated with minimum susceptibility
to syneresis (STS). The control bio-yoghurt had the lowest WHC and the highest STS. The maximum L. delbrueckii  subsp. bulgaricus and B. infantis counts were found in fresh and stored bio-yoghurt made with TGase when added after heat treatment of milk with pre-incubation at 50^oC/1h (A2). This increased counts of B. infantis throughout storage period and their populations remained above 10⁷cfu/g. The higher scores for all sensorial properties were recorded for  bio-yoghurt made with TGase added after heat treatment of milk (A1, A2), while the lowest scores were given for the control. Generally, bio-yoghurt of acceptable quality can be made from heated milk (92^oC/10min) followed by treatment with TGase (0.07%) with activation of the enzyme at 50^oC/1h before fermentation.