The new policy will require all VFL, VFLW and NAB league players and staff to receive both jabs ahead of the start of the 2022 seasons.
Unlike the fully professional AFL, the majority of people involved in these competitions are part-time and casual.
They will need to have their first dose by November 24 and are required to be fully vaccinated by December 22.
The AFL is also urging the SANFL and WAFL competitions to adopt similar policies in South Australia and Western Australia.
Players and staff in Victoria who choose not to be vaccinated will be barred from training and playing.
Like the AFL and AFLW programs, clubs will ultimately determine what happens to players who are adamant on not getting vaccinated.
"Our policy delivers on our commitment to be protect our players, staff and the wider community across many levels of football, so we can once again unite as families, as friends, as work colleagues, as teammates, as supporters, as communities, as one," AFL football executive Andrew Dillon said on Wednesday.
"State-league football and the talent pathway programs play an integral part in the football ecosystem that supports the elite AFL and AFLW competitions and the AFL shares responsibility to minimise the risk of exposure of COVID-19 across these levels of football."
"We will continue to work with all clubs, players and staff to provide the support and guidance through this evolving situation."
The status of community leagues will remain unchanged and local clubs and competitions will need to follow government health advice.
It comes after Carlton defender Liam Jones elected to retire from his AFL career, aged 30, rather than be vaccinated against COVID-19.
The call will cost him up to $500,000 after the former forward reinventing himself as a key part of the Blues' backline.
Jones was the first men's player to officially resist the vaccine mandate, but Adelaide AFLW premiership defender Deni Varnhagen has been moved to the Crows' inactive list for the upcoming season.
Varnhagen works as a registered nurse and has been outspoken in her anti-vaccination position, giving interviews at protests and posting about "global freedom rallies" on social media.
Jones' former Carlton teammate Levi Casboult is reportedly weighing up not getting vaccinated, which would cost him a shot at joining the Gold Coast Suns after he was delisted by the Blues in August.
AAP with The Project.