The purpose of this paper is to assess the competitiveness of science and technology innovation (STI) in national center cities in China (NCCC), identify the factors that limit their STI competitiveness, and propose ways to enhance their competitiveness. Firstly, a four-level evaluation index system is created by selecting 13 elements and 60 indicators from five STI dimensions: foundation, environment, input, output, and performance. Then, we use entropy-weighted TOPSIS, systematic cluster analysis, and geo-detector to conduct a comprehensive evaluation and cluster comparison analysis in the dimensions of time and space, and identify the driving forces and interactions. Our study reveals significant differences in the STI competitiveness of the nine NCCCs and their five subsystems. It also shows that the STI competitiveness of the old first-tier NCCCs in the developed regions of eastern China is generally higher than that of the new first-tier NCCCs in the less developed regions of central and western China. The suggestions proposed in this paper, such as enhancing the “head-wolf effect,” following the “city-by-city approach,” and promoting synergistic development, will not only help the NCCCs to become top-notch global STI centers but also serve as a reference for cities in other countries seeking to boost their STI competitiveness.