Zika virus (ZIKV) is an emerging pathogen with no approved therapeutics and only limited diagnostics available. To address this gap, six mouse single-chain antibodies (scFvs) to ZIKV envelope (E) protein were isolated rapidly and efficiently from a ribosome-displayed antibody library constructed from the spleens of five immunized mice. Methodology/Results In this report, we have generated a panel of mouse scFvs to ZIKV E protein using ribosome display. The six scFvs demonstrated no cross-reactivity with DENV2 NGC envelope protein, suggesting specificity for ZIKV E protein. These scFvs showed differences in their affinity: two (scFv45-3, scFv63-1) of them were dominant after four rounds of panning, and showed higher affinity (an apparent Kd values from 19 to 27 nM) than the other four (scFv5-1, scFv7- 2, scFv38-1, and scFv51-2). All six scFvs showed ZIKV-neutralizing activity in the plaque reduction neutralization test (PRNT) assay and their neutralizing activity was positively correlated with their affinities. Conclusions/Significance The scFvs (45-3 and 63-2) with highest affinity may have dual utility as diagnostics capable of recognizing ZIKV E subtypes and may be further developed to treat ZIKV infection. Our approach has the added advantage of generating Fc receptor-deficient antibodies, minimizing concern of antibody-dependent enhancement (ADE) of infection.