The river originates in the territory of Bad Großpertholz (Austria), then it crosses the Austrian-Czech border and flows through Pohorská Ves, then it returns to Austria and flows through Sankt Martin, Weitra and Unserfrau-Altweitra before it creates the Austrian-Czech border between the territories of Gmünd and České Velenice.

A small water reservoir, Kořensko, is built in the area of the confluence of the Lužnice and Vltava.

[6] A long section of the river forms the axis of the Třeboňsko Protected Landscape Area.

Within this area, the nature reserve of Horní Lužnice is delimited in the immediate vicinity of a 16-km long stretch of the river.

[6][7] The riverbed in the section between Veselí nad Lužnicí (where Třeboňsko PLA ends) to the mouth of the river is protected as Lužnice Nature Monument with an area of 432.2 ha (1,068 acres).

The reason for protection is the occurrence of rare and endangered species, especially thick shelled river mussel, weatherfish and Eurasian otter.

[11] At the village of Stará Hlína (part of Třeboň), the river is spanned by a stone inundation bridge.