This paper reports on application of ʟ-tryptophan modified magnetite nanospheres (TryMNSs) as a novel fluorescent chiral selector for selective and sensitive determination of ᴅ-phenylglycine (ᴅ-Phy) in the presence of its enantiomer (i.e. ʟ-phenylglycine, ʟ-Phy). The results showed that the nanospheres could be used as a “turn- on” fluorescence nanosensor based on enhancement of its fluorescence peak intensity in the presence of ᴅ-Phy. Unlike its enantiomer, ʟ-Phy did not affect the nanosensor fluorescence behaviour suggesting that using the synthesized nanospheres selective determination of ᴅ-Phy in the presence of ʟ-Phy was achievable. The enhancing effect can be attributed to the initial formation of hydrogen-bonded complex followed by π–π interaction between the aromatic tryptophan moieties and ᴅ-Phy. After various affecting factors optimizing, the results showed that using the enhancing effect, selective and sensitive determination of ᴅ-Phy in the presence of ʟ-Phy in the concentration range of 0.5–200.0 mg L −1 with a detection limit as low as 190 ng mL −1 was achievable.