Eleven return journeys will be offered each day between Kirkcaldy and Portobello, along with a shuttle bus link to Edinburgh city centre. Operated by Stagecoach, the 130-seat craft will take 40 minutes for the round trip and tickets will be priced at £4.50.
The £300,000 trial from 16 to 28 July coincides with the week-long closure of the Forth rail bridge for engineering work. If successful, a permanent service could follow to relieve congestion on the Forth Road Bridge.
Stagecoach chief executive Brian Souter remarked: "The Forth estuary is an underused resource and we are looking forward to giving people an opportunity to see the benefits this technology could bring to the region's communities, economy and public transport system."
The trial was originally planned for September last year but it was delayed when Scottish Natural Heritage raised environmental concerns about the scheme.