The Czechs, who became the first nation to retain the Davis Cup since Spain in 2009, lifted the trophy after Stepanek's 6-3, 6-1, 6-1 victory amid wild cheers by several hundred visiting fans in the imposing Kombank Arena.
Djokovic ensured the final went to the wire by beating Tomas Berdych 6-4, 7-6(5), 6-2 to level the tie at 2-2 but there was to be no repeat of Serbia's epic 3-2 final victory over France three years ago.
The world number two did not have the right support cast this time round with Janko Tipsarevic sidelined due to a foot injury and Viktor Troicki banned for missing a blood test in April.
The 117th-ranked Lajovic was thrown into the fray and had 17,000 noisy Serbian fans on their feet after he broke Stepanek, a former top-10 player now ranked 44th, in the opening game.
But that turned out to be the high point for the 23-year-old as his opponent highlighted the gulf in class between the two players.
"The match was the same (as last year's final), under the biggest possible pressure that can be," Stepanek, who beat Spain's Nicolas Almagro in the decisive rubber 12 months ago, said in a courtside interview.
"I knew that I would want to use the experience from last year's final. I controlled the match apart from the first game and I think I played in my best form ever this weekend.
"Of course, it is more difficult to defend the title, but I think we used the experience from last year and we earned it."
DESPAIRING BENCH
Stepanek combined his baseline and volleying skills to good effect, running Lajovic ragged with stinging forehands to carve out easy net points as the despairing Serbian bench watched on helplessly.
Earlier in the day, Djokovic came out on top against world number seven Berdych after a titanic tussle in the opening two sets.
He converted his 10th break point in the final game of the first, clinched a rollercoaster tiebreak in the second and wrapped up the third for the loss of only two games.
Djokovic blew kisses to the crowd and asked them to stay and support Lajovic's effort to punch above his weight, but it all ended in tears for Serbia as the stadium started to empty long before the 34-year-old Stepanek sealed victory with a searing smash down the line.
The result focused attention on the decision to leave Djokovic out of Saturday's doubles, which the Czechs won in straight sets, as well as naming Lajovic for the final singles instead of the more experienced and hard-hitting Ilija Bozoljac.
"We only had so many fit and eligible players to choose from and although I must take responsibility for the outcome as the decision maker, I have no regrets about the team selection in any of the rubbers," Serbia captain Bogdan Obradovic told a news conference.
"Missing two second-choice players was too big a blow and we just couldn't do more although we tried to cope with the tough situation as best we could. The positive thing is that we have expanded our Davis Cup team and we will keep the faith in our new arrivals."
Image: (L-R) Radek Stepanek, Lukas Rosol (team captain), Vladimir Safarik, Tomas Berdych and Jan Hayek of Czech Republic hold the winners trophy aloft after a 3-2 victory against Serbia.
Photograph: Srdjan Stevanovic/Getty Images