A digital microfluidic biochip (DMFB) is an attractive technology platform for revolutionizing immunoassays, clinical diagnostics, drug discovery, DNA sequencing, and other laboratory procedures in biochemistry. In most of these applications, real-time polymerase chain reaction (PCR) is an indispensable step for amplifying specific DNA segments. To reduce the reaction time to meet the requirement of “real-time” applications, multiplexed PCR is widely utilized. In recent years, three-dimensional (3D) DMFBs that integrate photodetectors (i.e., cyberphysical DMFBs) have been developed, which offer the benefits of smaller size, higher sensitivity, and faster result generations. However, current DMFB design methods target optimization in only two dimensions, thus ignoring the 3D two-layer structure of a DMFB. Furthermore, these techniques ignore practical constraints related to the interference between on-chip device pairs, the performance-critical PCR thermal loop, and the physical size of devices. Moreover, some practical issues in real scenarios are not stressed (e.g., the avoidance of the cross-contamination for multiplexed PCR). In this article, we describe an optimization solution for a 3D DMFB and present a three-stage algorithm to realize a compact 3D PCR chip layout, which includes: (i) PCR thermal-loop optimization, (ii) 3D global placement based on Strong-Push-Weak-Pull (SPWP) model, and (iii) constraint-aware legalization. To avoid cross-contamination between different DNA samples, we also propose a Minimum-Cost-Maximum-Flow-based (MCMF-based) method for reservoir assignment. Simulation results for four laboratory protocols demonstrate that the proposed approach is effective for the design and optimization of a 3D chip for multiplexed real-time PCR.