Biosensor is an analytical device to detect the biomolecules assisted by the transducer and physicochemical detector. A good biosensor is expecting to be with low cost, easy to perform and identify the results without prior experience. In addition, a good biosensor has two main key characteristics such as sensitivity and specificity; these are mainly determined by the affinity of biomolecules with the assistance of sensing system. Microfluidic-based lab-on-chip is one of the fast growing technologies in the field of biosensor bring the positive characteristics with a fast delivery set-up. On the other hand, gold nanoparticle (GNP) is the powerful tool to enhance the biomolecular detection with higher sensitivity and it has been proved for the effective applications with different sensors. In this review, we discussed the applications of microfluidic-based delivery and GNP for biosensing with the new level of developments, which elevate a step ahead.