Magnetic nanoparticle based bioseparation in microfluidics is a multiphysics phenomenon that involves interplay of various parameters. The ability to understand the dynamics of these parameters is a prerequisite for designing and developing more efficient magnetic cell/bio-particle separation systems. Therefore, in this work proof-of-concept experiments are combined with advanced numerical simulation to design and optimize the capturing process of magnetic nanoparticles responsible for efficient microfluidic bioseparation. A low cost generic microfluidic platform was developed using a novel micromolding method that can be done without a clean room techniques and at much lower cost and time. Parametric analysis using both experiments and theoretical predictions were performed. It was found that flow rate and magnetic field strength greatly influence the transport of magnetic nanoparticles in the microchannel and control the capturing efficiency. The results from mathematical model agree very well with experiments. The model further demonstrated that a 12% increase in capturing efficiency can be achieved by introducing of iron-grooved bar in the microfluidic setup that resulted in increase in magnetic field gradient. The numerical simulations were helpful in testing and optimizing key design parameters. Overall, this work demonstrated that a simple low cost experimental proof-of-concept setup can be synchronized with advanced numerical simulation not only to enhance the functional performance of magneto-fluidic capturing systems but also to efficiently design and develop microfluidic bioseparation systems for biomedical applications.