The Olympic javelin champion from Beijing 2008 and London 2012 won with a second round effort of 63.69m, her best throw for 11 months, as track and field events made a competitive return to the Czech Republic following the easing of coronavirus restrictions.
The 38-year-old got the better of 2018 European silver medallist Nikola Ogrodnikova whose best throw was 62.92m.
"I'm definitely happy with the performance. I said I wanted to throw over 60 metres and I did it." - Barbora Spotakova speaking to Czech TV
Spotakova and Stanek start season in style
With sport getting back up and running following the outbreak of COVID-19, the Czech Republic welcomed local athletes to Kladno, Kolin and Plzen for a series of Back on the Track meets.
Spotakova told Olympic Channel that her dream of winning a fourth medal at consecutive Olympic Games in Tokyo was "not open" after a struggle for form and fitness since the birth of her second child in 2018.
She faced a stern test on her seasonal debut, with Ogrodnikova improving her personal best to 67.40m last year.
The 29-year-old has also recently linked up with Spotakova's former coach, three-time Olympic champion Jan Zelezny.
Spotakova took the early lead with 57.31m after Ogrodnikova sent her spear out of bounds.
Ogrodnikova did the same in round two before her rival recorded 63.69m, her best distance since the Lausanne Diamond League meeting last July.
That mark would have been good enough for fifth in last year's World Championship final where Spotakova finished ninth.
Ogrodnikova hit back in round three with 62.92m, but she was unable to get any closer to the lead.
They both had good throws in the final round, Ogrodnikova with 62.52m and Spotakova 62.81m as the double Olympic gold medallist opened the season with a win.
Speaking to Czech TV afterwards, Spotakova said, "I was nervous as before every season. The first meet is always special and I'm glad that it finally took place this year. I enjoyed the conditions and I think I can be satisfied."
There was also an impressive performance in the men's javelin as 2017 world silver medallist Jakub Vadlejch took victory with an opening throw of 84.31m.
His Rio 2016 team-mate Tomas Stanek also made a winning start to the campaign.
Stanek, who finished fourth in the shot put at the 2017 World Championships, threw 21.13m in the second round to secure the win.
There was a personal best for Marek Barta in the men's discus.
The former Virginia Tech University star took victory with a throw of 62.95m.
Reigning three-time world indoor 400m champion Pavel Maslak had to settle for third in the men's 300m as Jan Jirka won in 32.82s.
Over in Kolin, the track highlight of the day came in the women's 100m hurdles.
Marketa Stolova led from start to finish with the 19-year-old clocking 13.62s, just six-hundredths of a second outside her personal best.
The high jump went to Bara Sajdokova who just missed out on the medals at the 2018 Buenos Aires YOG.
The 18-year-old cleared 1.81m at the first attempt, just as she had at her previous two heights, before failing three times at 1.85m.