It will be the second domestic route launched by the budget airline this year, after Bangkok-Krabi in January.
The airline plans to introduce an additional destination in the Northeast, as yet unnamed, in coming months, said an airline source.
The Northeast has the highest growth for an air travel market in Thailand. Nok Air claims a dominant market share in the region and is determined to raise its presence.
Nok Air serves seven provinces in the region — Udon Thani, Loei, Ubon Ratchathani, Nakhon Phanom, Sakhon Nakhon, Buri Ram and Roi Et — more than any other airline.
Khon Kaen is the commercial, administrative, and educational centre of the Northeast, often used by travellers as a base for visiting many parts of upper Isan, said the airline.
Nok Air will deploy a Boeing 737-800 with 189 seats for the Khon Kaen route, with promotional fares starting from 737 baht net one way.
Separately, FMG, the operator of Munich airport, has strengthened its partnership with Airports of Thailand (AoT).
The accord calls for a long-term continuation of the partnership between the two airport operating firms, which dates back to December 2004 when the Bavarian firm helped AoT commission Bangkok's Suvarnabhumi airport.
The agreement also envisages extending the scope of their joint activities to include consulting and infrastructure projects in Asia.
From December 2004 to September 2006, FMG's experts provided support to commission the new Bangkok airport at its new location, within the framework of an Operational Readiness and Airport Transfer project.
In December 2009, Munich airport and the state-controlled AoT signed a pact for a regular exchange of information and cooperation in operational processes, marketing, training and innovation management.