Fluorocarbon oils are uniquely suited for many biomedical applications due to their inert, bioorthogonal properties. In order to interface fluorocarbon oils with biological systems, non-ionic fluorosurfactants are necessary. However, there is a paucity of non-ionic fluorosurfactants with low interfacial tension (IFT) to stabilize fluorocarbon phases in aqueous environments (such as oil-in-water emulsions). We developed non-ionic fluorosurfactants composed of a polyethylene glycol (PEG) segment covalently bonded to a flexible perfluoropolyether (PFPE) segment that confer low IFTs between a fluorocarbon oil (HFE-7700) and water. The synthesis of a panel of surfactants spanning a molecular weight range of 0.64–66 kDa with various hydrophilic-lipophilic balances allowed for identification of minimal IFTs, ranging from 1.4 to 17.8 mN m⁻¹. The majority of these custom fluorosurfactants display poor solubility in water, allowing their co-introduction with fluorocarbon oils and minimal leaching. We applied the PEG₅PFPE₁ surfactant for mechanical force measurements in zebrafish, enabling exceptional sensitivity.