Multiple displacement amplification (MDA) has been widely used in biological and medical fields as a simple, stable and high-fidelity whole-genome amplification (WGA) method. From the reaction itself, many factors may influence the amplification quality of MDA. In this study, we analyzed MDA under different conditions of template input, amplification time, Mg²⁺ concentration, DNA denaturation methods and amplification temperature, and assessed their effects on the performance of MDA. We found that the use of different DNA denaturation methods before isothermal incubation can influence the amplification speed of MDA, which is represented as the reciprocal of the total time of the amplification reaction. Analyzed using next-generation sequencing, MDA coverage uniformity was found to be correlated with the amplification temperature. An appropriately high incubation temperature leads to more even coverage uniformity. Based on the results of this study, practical advice is offered to researchers to acquire better MDA performance.