Since perfluorooctane sulfonate (PFOS) had been detected in eggs of seabirds and fish, toxicity of waterborne PFOS to embryonic development of zebrafish (Danio rerio) was investigated. However, because assessment of effects by use of dietary exposure of adults is time-consuming and expensive, a study was conducted to compare effects on embryos via nanoinjection and waterborne exposure. Nanoinjection, in which small amounts of chemicals are injected into developing eggs, was used to incorporate PFOS into the yolk sac of embryos of zebrafish. Effects of PFOS during the period of development of the embryo were assessed within 96 h post-fertilization (hpf). PFOS significantly retarded development of embryos of zebrafish and resulted in abnormalities as well as lethality of developing embryos. Both methods of exposure, waterborne and nanoinjection, resulted in similar dose-dependent effects. Some sublethal effects, such as edema at 48 hpf, delayed hatching, and curvature of the spine was observed after 72 hpf. In ovo, nanoinjection was deemed to be an accurate method of exposure for controlling the actual internal dose for study of adverse effects, which closely mimicked responses to waterborne exposure of zebrafish embryo to PFOS.