With the widening application of graphene oxide nanosheets (GONS), their safety has attracted much attention. Secretions from aquatic organisms are ubiquitous in natural water, but the effects of secretions on the characteristics and toxicity of GONS remain largely unknown. To help fill this knowledge gap, we characterized the GONS with biological secretions (GOBS) and the associated changes in apparent toxicity. Small organic molecules, proteins, nucleotides and mucopolysaccharides from secretions in zebrafish culture water bound to GONS. Compared with GONS, GOBS showed special nanoplate topography with thicknesses of approximately 10 nm and lateral lengths ranging from 19.5 to 282 nm. GOBS with smaller lateral sizes exhibited more negative surface charges and lower aggregation state than GONS. Furthermore, GOBS triggered higher toxicity than GONS, such as death, malformation, upregulation of β-galactosidase and loss in mitochondrial membrane potential of zebrafish embryos. The well-dispersive GOBS covered embryos, inhibiting oxygen and ion exchange; these phenomena were the specific mechanisms of the adverse effects. In future work, the acquired natural coatings on nanomaterials should be paid much attention in nanotoxicology, especially for the relationships among topography, aggregation state, and toxicity.