French naturalist Francis de Castelnau described this species as Pseudoambassis macleayi in 1878, from specimens collected from the Norman River near the Gulf of Carpentaria in northern Australia.

[7] The species was subsequently returned to Ambassis in 1989 by J. R. Paxton, D. F. Hoese, G. R. Allen, and J. E. Hanley in the Zoological Catalogue of Australia.

[10] A. macleayi may be differentiated from other fish of the genus Ambassis by the relatively deep body, high gill raker count, interrupted lateral line and the dark spot at the base of the pectoral fins.

[10] In New Guinea, the fish is found in the trans-Fly region,[1] a lowland area of savanna and grasslands in the south of the island.

[12] Preferred habitat consists of streams and swamps, tending towards muddy lagoons at the end of the dry season to much further afield by the end of the wet season including lowland lagoons and the main channel of rivers and streams in the escarpments surrounding the Gulf of Carpentaria.

[10] It is typically found in heavily vegetated habitats with macrophytes, woody debris and leaf-litter and rarely at a depth of more than 1 m (3 ft).

[13] Ambassis macleayi is a moderately fecund fish with large females producing up to around 2,300 small (0.3 mm) eggs which expand on contact with water.

[10] A. macleayi is an obligate plant spawner,[14]: 178  with the eggs adhering to aquatic vegetation as they descend through the water column.

[15] A. macleayi is non-aggressive and suitable for larger community tanks and coexists well with other fish that are not small enough to be considered prey.

[17] Occasional live feed, such as brine shrimp or daphnia, is recommended by the Australia New Guinea Fishes Association.

[16] The Queensland Government recommends Macleay's glassfish for mosquito control in dams within the Gulf of Carpentaria drainage division.