WASHINGTON—The Justice Department is gearing up for an antitrust investigation of Alphabet Inc.’s Google, a move that could present a major new layer of regulatory scrutiny for the search giant, according to people familiar with the matter.

The department’s antitrust division in recent weeks has been laying the groundwork for the probe, the people said. The Federal Trade Commission, which shares antitrust authority with the department, previously conducted a broad investigation of Google but closed it in 2013 without taking action, though Google made some voluntary changes to certain business practices.

The FTC and the department have been in talks recently on who would oversee any new antitrust investigation of a leading U.S. tech giant, and the commission agreed to give the Justice Department jurisdiction over Google, the people said.

With turf now settled, the department is preparing to closely examine Google’s business practices related to its search and other businesses, the people said.

It couldn’t immediately be learned whether Google has been contacted by the department. Third-party critics of the search giant, however, already have been in contact with Justice Department officials, some of the people familiar with the matter said.