We present a detailed numerical analysis of electrophoresis induced concentration of a bio-analyte facilitated by temperature gradient focusing (TGF) in a phosphate buffer solution via Joule heating inside a converging–diverging microchannel. The purpose is to study the effects of frequency of AC field and channel width variation on the concentration of target analyte. We tune the buffer viscosity, conductivity, and electrophoretic mobility of the analyte such that the electrophoretic velocity of the analyte locally balances the electroosmotic flow (EOF) of the buffer, resulting in a local build-up of the analyte concentration in a target region. An AC field is superimposed on the applied DC field within the microchannel in such a way that the back pressure effect is minimized, resulting in minimum dispersion and high concentration of the target analyte. Axial transport of fluorescein-Na in the phosphate buffer solution is controlled by inducing temperature gradient through Joule heating. The technique leverages the fact that the buffer’s ionic strength and viscosity depend on temperature, which in turn guides the analyte transport. A numerical model is proposed and a finite element-based solution of the coupled electric field, mass, momentum, energy, and species transport equations are carried out. Simulation predict peak of 670-fold concentration of fluorescein-Na is achieved. The peak concentration is found to increase sharply as the channel throat width decreases, while the axial spread of concentrated analyte increases at lower frequency of AC field. The results of the work may improve the design of micro concentrator.