Sanctuary began life as an eight-episode webisode series released online in early 2007, and its success convinced Syfy to buy the broadcast rights and re-stage it as a full 13-episode television season.

The series' first two TV episodes, 'Sanctuary for All' and 'Fata Morgana', were combinations and rewrites of the original eight webisodes, with Amanda Tapping and the entire original web-series cast making the transition to television.

Sanctuary is filmed almost entirely using green screen technology, with no elaborate physical sets built; environments are instead created with CGI, though a few first-season episodes like 'Kush' and 'Requiem' used practical sets, including an actual aircraft fuselage.

The webisodes were shot from January 3 to January 31, 2007, at Bridge Studios, and the first television season, costing an estimated CA$21 million, was filmed in Burnaby, British Columbia.

The episode 'Instinct' was filmed in a distinct, Cloverfield-esque handheld style, differing from the show's usual visual approach.

Sanctuary was the first television series in North America to use the RED camera system exclusively, recording straight to hard drive at resolutions up to four times standard HD, which gave the visual effects and post-production teams immediate access to each day's footage.

Christopher Heyerdahl played two different characters in the series: the taciturn Neanderthal-like Sanctuary employee credited as 'Bigfoot' and John Druitt, a member of 'The Five' who becomes Jack the Ripper.

Former Stargate Atlantis actor Paul McGillion joined the second season to play Wexford, a character he had already portrayed in the original web series, while former Stargate SG-1 actor Michael Shanks guest-starred as Jimmy in the episode 'Penance'.

Amanda Tapping served as an executive producer on the show but stated in an interview that she received no extra pay for that role, since executive producer salaries went toward funding the sets, as the series lacked studio backing.

The pilot episode 'Sanctuary for All' drew more than 3 million viewers and received the highest Nielsen rating for a Syfy original series premiere since Eureka debuted in July 2006, making it the number one cable program among adults 25–54 that week.