The first radiation of eukaryotic life in Earth history began at least by the late Paleoproterozoic as evidenced by complex organic-walled microfossils from North China, India and Australia. However, among these oldest eukaryotic fossils, the spinose Tappania plana Yin, 1997, which has been reported from the late Paleoproterozoic to early Mesoproterozoic sediments worldwide, is strangely absent in the well-known microfossil assemblages from the coeval strata in the Yanliao Basin, North China. Here we report the new finding of T. plana and associated microfossils from similar to 1.64 Ga Chuanlinggou Formation in the eastern Yanliao Basin. T. plana occurs in the assemblage from the upper Chuanlinggou Formation and exhibits typical irregularly distributed tubular processes and neck-like extensions. The assemblage is dominated by a wide variety of filamentous microfossils likely representing cyanobacterial or other bacterial sheaths and trichomes, and includes 21 species (belonging to 10 genera) and 3 unnamed forms. In contrast, the assemblage from the lower Chuanlinggou Formation is composed of only simple spheroidal (Leiosphaeridia) and fusiform (Schizofusa) morphotypes, distinct from those of the upper assemblage. This taxonomic discrepancy likely resulted from different paleoenvironments. Overall, the occurrence of T. plana in the Yanliao Basin expands its paleographic distribution and further reinforces its stratigraphic potential as an index fossil for the late Paleoproterozoic and early Mesoproterozoic strata. Furthermore, the microfossil assemblages reported here document a microbiota overwhelmingly dominated by prokaryotic organisms, which is consistent with the hypothesis that eukaryotes likely constituted as marginal groups in ancient ecosystems during mid-Proterozoic.