According to Official Guinness Records,
The world's oldest botanical garden, which remains on its original site, is the Orto Botanico in Padua, Italy, which was created in 1545. The original design of a circular central plot (symbolising the world), surrounded by a ring of water remains, as does its original purpose - a place for scientific research.The oldest botanical garden ever is the Botanical Garden of Pisa, Italy, which was founded in 1544, but was relocated twice, before settling on its present site since 1591.
For a complete list of 2002 records, please visit 2002 Guinness Records in Italy.