Kefir grains represent a symbiotic association group of yeasts, lactic acid bacteria and acetic acid bacteria within an exopolysaccharide and protein matrix known as kefiran. The mechanism of growth of a biomass of kefir after successive fermentations and optimal conditions is not well understood yet. Biomass growth kinetics were determined to evaluate the effects of temperatures (10 °C to 40 °C) and different substrates, such as monosaccharides (fructose, galactose, glucose), disaccharides (lactose, saccharose) and polysaccharides (Agave angustifolia fructans) at 2%, in reconstituted nonfat milk powder at 10% (w/v) and inoculated with 2% of milk kefir grain (105 CFU/g), after determining the pH kinetics. The best conditions of temperature and substrates were 20 °C and fructans and galactose. An increase in cells, grain sizes and a change in the morphology of the granules with the best substrates were observed using environmental scanning electron microscopy, confocal laser scanning microscopy and Image Digital Analysis (IDA). Kefir grains with agave fructans as their carbon source showed the higher fractal dimension (2.380), related to a greater co-aggregation ability of LAB and yeasts, and increase the formation of exopolysaccharides and the size of the kefir grains, which opens new application possibilities for the use of branched fructans as a substrate for the fermentation of milk kefir grains for the enhancement of cellular biomasses and exopolysaccharide production, as well as IDA as a characterization tool.