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We have shown that Au nanoparticles (Au NPs) pearl chain could be formed consistently between microelectrodes by combining microspotting and DEP technologies. Experimental results on varying the Au particle size and dielectrophoretic (DEP) parameters, including voltage and frequency, are reported in this paper to explore the critical parameters in controlling the Pearl Chain Formation (PCF) process between microelectrodes. PCF was observed from 10 kHz to 5 MHz for 100 nm Au NPs, and 100 kHz to 10 MHz for 10 nm Au NPs. Variations in formation rate were detected when the applied voltage and particle size varied. With higher voltage, pearl chain began to form at higher rate and the formation time decreased. The optimum frequency of the Au NPs PCF shifted to higher frequency region when the particle size decreased. Theoretical analysis was carried out by applying the theories of DEP force and AC electrokinetics to explain the observations with the DEP frequency ranging from 10 Hz to 10 MHz. Finally, Au nanoparticle chains formed between the microelectrodes were shown to vary in resistance when environmental temperature was changed, indicating that these Au particle sensors could potentially be used to sense temperature and other thermal-based physical phenomena.