Polymerase chain reaction (PCR) has become a fundamental technique in molecular biology. Nonetheless, further improvements of the existing protocols are required to broaden the applicability of PCR for routine diagnostic purposes, to enhance the specificity and the yield of PCRs as well as to reduce the costs for high-throughput applications. One known problem typically reported in PCR experiments is the poor amplification of GC-rich DNA sequences. Here we designed and tested a novel effective and low-cost PCR enhancer, a concentration-dependent combination of betaine, dithiothreitol, and dimethyl sulfoxide that broadly enhanced the quantitative and/or qualitative output of PCRs. Additionally, we showed that the performances of this enhancer mix are comparable to those of commercially available PCR additives and highly effective with different DNA polymerases. Thus, we propose the routine application of this PCR enhancer mix for low- and high-throughput experiments.