Decimetre-scale carbonaceous macrofossils from the Mesoproterozoic Gaoyuzhuang Formation in the Yanshan Range are known as the current oldest unambiguous evidence of macroscopic multicellular eukaryotes. Here, we reported a new SIMS zircon age of 1588.8 ± 6.5 Ma from a volcanic tuff in the Qianxi County of Hebei Province, about 11 m above the macrofossil’s horizon. This new age provides a direct age constraint on the macroscopic eukaryotic fossils from the Gaoyuzhuang Formation. It indicates that macroscopic life with the moderate diversity and certain morphological complexity had already evolved at the beginning of the Mesoproterozoic, and implies a possibility of discovering macroscopic eukaryotes in earlier rocks. This study also calls for a stratigraphic framework to integrate biological and environmental studies in different regions for a better understanding of the evolution of multicellular organisms and environmental change during this important period.