The key role of calcium in whey protein fouling behavior is well known in plate heat exchangers (PHEs), as it affects both the thermal denaturation of proteins and deposition reaction on the surface. However, the complex flow pattern and the closed configuration of PHEs make it hard to investigate this phenomenon in situ. In this paper, a microchannel benchtop fouling device was designed, making it possible to achieve a similar temperature profile to that performed in PHEs but in a laminar regime. A 3D simulation was developed to predict the thermal denaturation of β-lactoglobulin (BLG) as well as its deposition in the microchannel using computational fluid dynamics (CFD). The thermodynamics and heat transfer of the numerical model was validated by experimentally measuring the bulk fluid temperature profile in the microchannel using fluorescence microscopy, where Pyrromethene 556 was used as a temperature indicator. Results revealed a quasi-linear relationship between the pre-exponential factor of deposition reaction and the calcium concentration, implying that the fouling was built in such a pattern that only one calcium ion per one BLG molecule is involved. The imaging of the fouling deposit in situ showed spherical structures of deposits at low calcium levels, while denser and more rod-like ones were found at higher calcium concentrations. The fouling behavior was found to follow a crystallization-like pattern with preference upon the previously fouled layer instead of clean stainless steel surface. These findings confirm the essential role of ionic calcium on the formation of fouling deposits by anchoring the denatured BLG protein upon the surface as well as strengthening the protein-protein interactions for fouling build-up.