In this work, Cu and Cu55Ni45 thin films with a thickness of 600 nm were firstly deposited on single crystal Si and Al2O3 ceramic substrates by magnetron sputtering respectively. Then, the thin film thermopiles composed of 200 pairs of in-series thermocouples were fabricated by microfabrication technology in 10 mm×10 mm substrate area. Finally, aluminum oxide layers were deposited by reactive magnetron sputtering as thermal resistance layers, with the help of hard mask. The different thickness of the aluminum oxide layer produces the cold and hot ends in the thin film thermopile, giving rise to a voltage under the irradiation of heat flux by the Seebeck effect, realizing the measurement of heat flux. The thin film thermopiles were analyzed and calibrated. The results show that in the Cu/Cu55Ni45 thermopiles deposited on the Si substrate and Al2O3 ceramic substrate, the roughness of the Cu films are 20 and 60 nm, the roughness of Cu55Ni45 films are 15 and 20 nm, the electrical resistance of thermopiles are 38.2 Ω and 2.83 kΩ, the sensitivity of thermopiles are 0.069 45 and 0.026 97 mV/(kW·m-2), respectively. The surface roughness of Cu/Cu55Ni45 thermopiles deposited on single crystal Si substrate and Al2O3 ceramic substrate with different surface roughness will be affected, resulting in the difference in electrical resistance of thin film thermopile. In addition, the output thermoelectric voltage exhibits a good linear relationship with heat flux.