An experimental method is demonstrated to enhance the refractive index sensitivity (RIS) of gold (Au) ultrathin films used in a localized surface plasmon resonance (LSPR) sensor as an optical transducer using sequential temperature treatment in fabrication processes. The Au ultrathin films were fabricated by the vacuum thermal evaporation technique to deposit Au nanoparticles on a glass substrate, followed by annealing. The ultrathin layer of Au had a thickness of 1.13 nm. Furthermore, two different annealing times were employed in two different treatment processes with annealing temperature of 400 °C. The first treatment involved longer annealing times (8 h, 12 h, 24 h, and 36 h) and a normal cooling process (cooling rate: 0.02 °C/s); the second treatment involved shorter annealing times (1 min, 5 min, 10 min, and 30 min) and a quenching process (cooling rate: 25 °C/s). The Au ultrathin films were spectroscopically evaluated based on their morphology and optical properties in the transmissive mode integrated in a self-assembled optofluidic. The highest RIS of the Au ultrathin films was obtained at 240.45 nm/RIU for the short annealing time-quenching treatment and 90.85 nm/RIU for the long annealing time-normal cooling treatment. This method demonstrates the great potential toward fabrication of Au ultrathin films based LSPR for liquids sensing.