MiR-155 plays a critical role in the formation of cancers and other diseases. In this study, the authors aimed to design and fabricate a biosensor based on cross-linking gold nanoparticles (AuNPs) aggregation for the detection and quantification of miR-155. Also, they intended to compare this method with SYBR Green real-time polymerase chain reaction (PCR). Primers for real-time PCR, and two thiolated capture probes for biosensor, complementary with miR-155, were designed. Citrate capped AuNPs (18.7 ± 3.6 nm) were synthesised and thiolated capture probes immobilised to AuNPs. The various concentrations of synthetic miR-155 were measured by this biosensor and real-time PCR method. Colorimetric changes were studied, and the calibration curves were plotted. Results showed the detection limit of 10 nM for the fabricated biosensor and real-time PCR. Also, eye detection using colour showed the weaker detection limit (1 µM), for this biosensor. MiR-133b as the non-complementary target could not cause a change in both colour and UV-visible spectrum. The increase in hydrodynamic diameter and negative zeta potential of AuNPs after the addition of probes verified the biosensor accurately fabricated. This fabricated biosensor could detect miR-155 simpler and faster than previous methods.