The advertisement, the second in two days, revived arguments between the two rivals over national security policy between the two during the primaries.
The advert reprised Clinton's comment during the primary battle that: "I know Senator McCain has a lifetime of experience that he will bring to the White House, and Senator Obama has a speech he gave in 2002."
"Hillary's right. John McCain for president," the advertisement concluded.
Al Jazeera's Rob Reynolds said Clinton would try to unify the party but also be sharply on the attack against John McCain and link him to policies of the Bush administration.
|Some Hillary Clinton supporters
Terry McAuliffe, Clinton's campaign chairman, told Al Jazeera that her speech would help win over her disgruntled supporters.
"Let's be honest, this was a long, tough primary campaign, Hillary got 18 million votes, she won all the big states. Everybody put their heart and soul into that.
"And it's going to take some people longer than others.
"But Hillary's arguments tonight and her speech I think is the beginning of the end.
"We're going to have a roll call so people can voice their support for Hillary Clinton, tomorrow night.
"And when that's all done, and then Bill Clinton speaks, I think we will have moved tremendously toward bringing those people over."
Polls show that despite Clinton's repeated public shows of support for Obama, many of her voters are still not likely to vote for her former rival.
One poll released on Monday said 30 per cent of Hillary supporters could vote for McCain instead of Obama.
However, Anna Cruz, a Democratic political consultant, told Al Jazeera that those figures were not entirely accurate.
"I think that the poll numbers we are seeing here in the United States don't reflect that 30 per cent are definitely with John McCain.
"We will see those voters that haven't come over so easily, they will trend and they will move toward Barack Obama.
"I can tell you this much right now, that senator Hillary Clinton and senator Barack Obama are far much more aligned with each other than John McCain will ever be with Democrats."
The next first lady?
Thousands of people are attending the four-day gathering in the US state of Colorado.
Delivering the keynote speech on Monday, Michelle Obama told the assembly that her husband represented typical American values.
"I come here as a wife who loves my husband and believes he will be an extraordinary president," she told the audience.
Barack Obama, campaigning in Iowa, also made an appearance via video screen as his two daughters crowded onto the Denver stage with his wife.
Obama promised his much-hyped nomination acceptance address to the convention on Thursday - the 45th anniversary of Martin Luther King Jr's "I Have a Dream" speech - would be "workmanlike", focusing on policy details over propaganda.