Nowadays, isothermal amplification-based biomolecular detection have gathered extensive interests due to its easy applicability toward various point-of-care testing (POCT) devices. It is a promising strategy which can successfully replace conventional polymerase chain reaction (PCR)-based assays. In this study, we utilized fluorescent copper nanoparticles (CuNPs) as our signaling materials for isothermal amplification-based assays due to their easy, simple, eco-friendly, and rapid synthesis compared with other fluorescent nanomaterials. In chapter 2, we devised a rapid and ultrasensitive microRNA (miRNA) detection strategy by employing target-assisted isothermal exponential amplification (TAIEA) combined with poly (thymine)-templated fluorescent CuNPs as signaling probes. The target miRNA hybridizes to a unimolecular template DNA and works as a primer for extension reaction to generate double-stranded product which contains two nicking endonuclease recognition sites. By simultaneous nicking and displacement reactions, exponential amplification generates poly (thymine) strands as final products, which are directly utilized for the synthesis of fluorescent CuNPs. As a result, our target miRNA was successfully detected as low as 0.27 fM around 1 hour of total analysis time. In chapter 3 and 4, we developed a novel rolling circle amplification (RCA) method coupled with poly (thymine)-templated fluorescent CuNPs for rapid and ultrasensitive target DNA detection and enzymatic assay. At first, ligation reaction is performed to generate circular template for initiating RCA reaction. Then, long and single-stranded DNA is generated by using phi 29 DNA polymerase and consuming dNTPs. Resultant product contains many repetitive poly (thymine) sequences which can be further utilized as templates for the synthesis of fluorescent CuNPs. Therefore, amplified fluorescence signal is obtained from resultant RCA product for ultrasensitive assay. As a result, our target DNA and enzyme were successfully detected around 2 hours of total analysis time as low as 0.49 aM and 0.01 U/mL, respectively.