Assessing preferential flow that occurs through small fractures or permeable connected pathways of other kinds is important to many processes but is difficult to determine, since most chemical tracers diffuse quickly enough from small flow channels that they appear to move more uniformly through the rock than they actually do. Conventional tracer and micro tracer experiments were conducted using two different synthetically single-fractured limestone core plugs to investigate non-isothermal-coupled heat and mass transfer in a single fracture-matrix system. Conventional Rhodamine B and suspension of micro particles based on melamine resin, Rhodamine B-marked size 1-µm tracer with constant concentration were injected at constant flow rate during non-isothermal flow-through experiments. Effluent tracer concentrations were measured using the fluorescence spectrometry technique. A numerical simulation model was used to determine effective thermal and physical properties involved in matrix-fracture transfers during conventional tracer injection. Numerical studies indicated that matrix permeability is the main controlling factor for tracer dispersion within the matrix. The results showed that tracer transfer from the fracture to the matrix at low injection rates is higher compared to that of higher injection rates. As the fracture provides a large transport pathway for micro particles compared to the surrounding matrix, advection causes the early breakthrough for micro particle transport through a single fracture-matrix system. When the conventional tracer has a long enough transit through the system to diffuse into the matrix, but the micro tracer does not, the micro tracer arrives first and the conventional tracer later, and the separation measures the degree of preferential flow.