'Purito' attacked in the final part of the Salita di Ello, a 3.25-km ascent at an average gradient of 7.4 percent, and held off a strong chasing group on the flat.
Rodriguez of Katusha came clear of Spanish compatriot Samuel Sanchez (Euskaltel Euskadi) to win by nine seconds, while Team Sky's Rigoberto Uran was third on the same time.
The Giro di Lombardia, the last 'Monument' of the European calendar, is a 251km race which this year began in Bergamo and finished in Lecco.
In claiming the 106th edition of the race Rodriguez became the first Spaniard in the race's history to take victory.
The result also means that the rider is sure to end the season as the world's number one-ranked cyclist on the UCI official standings.
Bradley Wiggins, the Tour de France champion, had held that position, but Rodriguez, who has also won La Fleche Wallonne, and achieved two top threes in the general classification of the Giro d'Italia and Vuelta a Espana this year, now trumps him on the rankings.
"This is the most important triumph of my whole career," Rodriguez was quoted as saying in a team statement.
"This was a perfect season. Today I was feeling in great shape: in fact I made my team mates work during all the crucial moments of the race.
"When I saw that all my rivals were tired and I felt so great, I realised I had a great chance to win. To tell the truth I thought that somebody could join me in that attack, but instead nobody could answer and that makes this victory even greater."
As expected, the brutal climb to the Muro di Sormano sorted the men from the boys.
The two-kilometre ascent at an average gradient of some 15 percent blew the peloton apart with Alberto Contador, Vincenzo Nibali and Rodriguez among those setting the pace.
World champion Philippe Gilbert crashed in the descent and pulled out.
"He's got some burns and cuts but nothing serious," his BMC team manager John Lelangue told RAI.
Nibali also hit the deck in the descent from the Madonna del Ghisallo but managed to get back on the bike and rejoin the peloton.
The Italian, one of the pre-race favourites with Contador, Rodriguez and Gilbert, had not fully recovered before the last ascent and did not figure in the finale.