Self-assembly is a mechanism that creates novel nanomaterials with amplified properties but with stability challenges. In this report, highly stable and biocompatible anionic zwitterionic diphenylalanine nanoparticles (ZFFNPs), which are novel for the literature, are demonstrated. ZFF was crosslinked with glutaraldehyde (GA) and self-assembled into nanoparticles in a self-assembly mechanism like that for diphenylalaninamide nanoparticles (FFANPs). Also, ZFFNPs were compared with FFANPs in aspects of morphology, surface charge, stability, and cytotoxicity. ZFFNPs demonstrate a spherical morphology and homogenicity like FFANPs, but while ZFFNPs have negative surface groups (carboxyl), FFANPs contain polar surface groups (amide). While ZFFNPs exhibit a high stability in extremely acidic environments (pH 3-5), AFFNPs show stability in a wide pH range (pH 4-10). Both NPs are nontoxic and biocompatible. These novel anionic ZFFNPs have great promise for potential utilization in biomedical applications. (C) 2017 Wiley Periodicals, Inc.