The Paleo- and Mesoproterozoic fossil record of multicellular eukaryotes is scarce but extremely important in studying the evolution of life in the Precambrian Era. Typically, fossils heralded as ‘multicellular eukaryotes’ that are older than the Ediacaran are met with controversy. To confront such debates, we investigate the chemical, isotopic, and molecular structural characteristics of organic matter from carbonaceous compressions in the 1630 Ma Tuanshanzi Formation in northern China, some of the earliest putative macroscopic multicellular eukaryote fossils (Zhu and Chen, 1995). The large-size and relative complexity of these fossils and similar remains from the 1.56 Ga Gaoyuzhuang Formation have led to their interpretation as eukaryotes. Raman spectral parameters give an estimated peak-metamorphic temperature TMax in the range of 202–286 °C, confirming the good preservation of the organic remains. Two-dimensional Raman maps of the carbonaceous compressions show ultrastructural variations that suggest diverse subcellular compounds being consistent with multicellular eukaryotes. The organic matter has carbon isotopic composition δ13Corg-SIMS between −45.3 and −32.2‰, and a branching index of carbon chain measured by the micro-FTIR spectral parameter R3/2 between 0.17 and 0.31. Together with their large-size and morphology, the isotopic, geochemical and ultrastructural data supports an interpretation of the Tuanshanzi Formation carbonaceous compressions as derived from phototrophic, multicellular eukaryotic algae. Our data support the early evolution of macroscopic multicellular eukaryotes in the sulfidic and low-oxygen conditions that prevailed in the Paleo- and Mesoproterozoic oceans.