Brown adipocytes function to maintain body temperature by heat production. However, direct measurement of heat production at a single cell level remains difficult. Here we developed a method to measure the temperature within primary cultured brown adipocytes using a cationic fluorescent polymeric thermometer. Placement of the thermometer within a matured brown adipocyte and a precursor cell enabled the detection of heat production following uncoupler treatment. The increase in the intracellular temperature due to stimulation with a mitochondrial uncoupler was higher in matured brown adipocytes than in precursor cells. Stimulation with a β-adrenergic receptor (β-AR) agonist, norepinephrine, raised the intracellular temperature of matured brown adipocytes to a level comparable to that observed after stimulation with a β3-AR-specific agonist, CL316.243. In contrast, neither β-AR agonist induced an intracellular temperature increase in precursor cells. Further, pretreatment of brown adipocytes with a β3-AR antagonist inhibited the norepinephrine-stimulated elevation of temperature. These results demonstrate that our novel method successfully determined the difference in intracellular temperature increase between matured brown adipocytes and precursor cells in response to stimulation by an uncoupler and β-AR agonists.