Fiber-optic distributed temperature sensing (FO-DTS) has been frequently applied for analyzing thermal patterns, including the identification of groundwater-surface water exchange fluxes across aquifer-river interfaces. However, the impacts of (a) seasonal variability in signal strength (given by the difference between groundwater and surface water temperatures) and (b) monitoring modes on the accuracy of FO-DTS surveys have not yet been determined. This study uses a well-investigated field site as model system for quantifying the accuracy and uncertainty of FO-DTS surveys in dependency of seasonal signal variation and monitoring mode. The analysis of the relationship between seasonal variability in signal strength and diurnal oscillations in end-member temperatures at the study site revealed that winter conditions, with substantially lower diurnal temperature oscillations, provide the highest temporal stability in signal strength. The choice of monitoring mode proved to have significant impact on the accuracy of FO-DTS surveys. The proposed two-way single-ended averaging of FO-DTS surveys had significant advantages compared to single-ended or double-ended surveys, with a higher accuracy in signal detection, in particular for small-scale temperature variations. Since FO-DTS surveys in two-way single-ended averaging mode were better suited for detecting the full complexity of spatial temperature patterns for the investigated aquifer-river interface, we recommend its wider application in similarly complex systems with small-scale thermal patterns.