Alkaline phosphatase (ALP) is a nonspecific phosphatase, and its interaction with substrates mainly depends on the recognition of phosphate groups on the substrate. Previous enzymatic research has focused mainly on the enzymatic reaction kinetics of the inorganic small molecule p-nitrophenol phosphate (pNPP) as a substrate, but its interaction with biomacromolecule substrates has not been reported. In current scientific research, ALP is often used for molecular cloning, such as removing the 5' termini of nucleic acids. However, no detailed reports on the interactions between ALP and these biomolecules have been published. We used microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) experiments to investigate the affinity of mutant ALP (S102L) from Escherichia coli for biomacromolecule substrates, including double-stranded DNA (dsDNA) and phosphoproteins. We found that S102L ALP has a strong affinity for dsDNA and β-casein. For the first time, the affinity of ALP for the substrate phosphate monoester has been proven to be significantly affected by the nature of its R group (ROP). In summary, we have explained the key factors involved in the interaction between ALP and biomacromolecule substrates from the perspective of affinity, which provides guidance in better understanding ALP.