Aptamers are promising affinity ligands with considerable applications, such as biosensors, disease diagnosis, therapy, etc. Characterization of aptamer-target binding is important in aptamer selection and aptamer applications. Microscale thermophoresis (MST) is an emerging optical technique for molecular interactions, which monitors fluorescence responses of fluorescent molecules in a microscopic temperature gradient. Harnessing merits in trace sample consumption, high speed, free separation, free immobilization, and ratiometric analysis, MST draws intense wide attention. MST is often applied for aptamer-target binding studies using fluorescently labeled aptamers. However, the MST signal is strongly dependent on fluorophore modifications at aptamers, which brings additional challenges and effects for MST analyzing aptamer affinity. Here, we systematically explored effects of fluorophore modifications (e.g., fluorophore types, fluorophore positions, etc.) of aptamer probes on MST characterizing aptamer-target interactions and identified gaps of MST analysis in aptamer affinity determination, taking aptamers against cadmium ions and aflatoxin B1 as two representatives. The same aptamers with different fluorophore modifications showed distinct MST signals in response magnitudes and signs as well as determined affinities, and some of them failed to respond to target binding and gave false affinity information in MST. A competitive MST method can be used to extract the affinity of unmodified aptamers, excluding effects of fluorophore modification. This work highlights that appropriate fluorophore modification is crucial in MST analysis of aptamer affinity, and caution is needed in MST applications, providing a basis for rational design of the MST method for the reliable molecular interaction study.